Effects of Mutation or Deletion

Table of contents

Wingless function in imaginal discs

Flies bearing a temperature sensitive allele of strawberry notch show a modest loss of wing margin tissue when raised at 23 degrees C. When such flies are also made heterozygous for a single copy loss of wingless, extensive loss of wing margin tissue is observed, suggesting a dominant synergistic interaction between wg and sno. In similar experiments, temperature sno combined with a single copy loss of vestigial also results in a dominant enhancement of wing margin defects; mutants exhibit extensive loss of wing margin tissue. Genetic combination of a weak allele of cut with a sno mutation shows extensive loss of wing margin tissue, suggesting a synergistic interaction between sno and cut. These results are reminiscent of the interaction of Notch with wg, vg and ct and further extablish that sno, like Notch, has a crucial role in the establishment of D/V boundary fate by participating in a common genetic pathway that regulates wing margin-specific genes. In addition to the wing margin defects, sno mutants also exhibit thickening of wing veins. This is likely to be a secondary consequence of defective wing pouch development caused by improper D/V boundary specification. This same phenotype can also be seen in some of the other D/V boundary genes, such as vestigial and Serrate (Majumdar, 1997).

Each of the somatic cell types of the gonad arises from mesodermal cells that constitute the embryonic gonad. The functions of the homeotic genes abdominal A and Abdominal B are both required for the development of gonadal precursors. Each plays a distinct role. abd A activity alone specifies anterior somatic gonadal precursor (SGP) fates, whereas abd A and Abd B act together to specify a posterior subpopulation of gonadal precursors. Once specified, gonadal precursors born within posterior parasegments move to the site of gonad formation. clift has been identified as a regulator of Drosophila gonadogenesis. When cloned, clift turned out to be identical to eyes absent. Mutations in clift abolish gonad formation and produce female sterility. Just as with abdominal A, clift is expressed within SGP as these cells first form, demonstrating that 9-12 cells are selected as SGP within each of three posterior parasegments at early stages in gonadogenesis. Using clift as a marker, it has been shown that the anteroposterior and dorsoventral position of the somatic gonadal precursor cells within a parasegment are established by the secreted growth factor Wingless, acting from the ectoderm, coupled with a gene regulatory hierarchy involving abd A within the mesoderm. While loss of wg abolishes gonadal precursors, ectopic expression expands the population such that most cells within lateral mesoderm adopt gonadal precursor fates. Initial dorsoventral positioning of somatic gonadal precursors relies on a regulatory cascade that establishes dorsal fates within the mesoderm. tinman appears to mediate the role of ectodermally expressed decapentaplegic; in tinman mutants few or no SGP cells are detected. clift expression is subsequently refined through negative regulation by bagpipe, a gene that specifies nearby visceral mesoderm. Thus, these studies identify essential regulators of gonadal precursor specification and differentiation and reveal novel aspects of the general mechanism whereby somatic gonadal cell fate is allocated within the mesoderm (Boyle, 1997).

Genetically mosaic flies were constructed which lack a functional dpp or wg gene in portions of their leg epidermis, and the leg cuticle was examined for defects. Although dpp has been shown to be transcribed both ventally and dorsally, virtually the only dpp-null clones that affect leg anatomy are those which reside dorsally. Conversely, wg-null clones only cause leg defects when they reside ventrally. Both findings are consistent with models of leg development in which the future tip of the leg is specified by an interaction between dpp and wg at the center of the leg disc. Null clones can cause mirror-image cuticular duplications, confined to individual leg segemnts. Double-ventral, mirror-image patterns are observed with dpp-null clones, and double-dorsal patterns with wg null clones. Clones that are doubly mutant (null for both dpp and wg) manifest reduced frequencies for both types of duplications. Duplications can include cells from surrounding non-mutant territory. Such nonautonomy implies that both genes are involved in positional signaling, not merely in the maintenance of cellular identities. However, neither gene product appears to function as a morphogen for the entire leg disc, since the effects of each gene's null clones are restricted to a discrete part of the circumference. Interestingly, the circumferential domains where dpp and wg are needed are complementary to one another (Held, 1996).

Dominant mutations provide invaluable tools for Drosophila geneticists. The dominant eye mutation Glazed (Gla), described by T. H. Morgan more than 50 years ago, has now been analyzed. Gla causes the loss of photoreceptor cells during pupal stages, in a process reminiscent of apoptosis, with a concomitant overproduction of eye pigment. Ommatidial bristles are missing in the anterior-ventral part where the Gla mutant phenotype is generally more pronounced. Most of the eye appears to consist of pigment cells since pigment granules are highly abundant over the entire surface. Pigment cell shape is predominantly rectangular, suggesting that most of the pigment cells have adopted a tertiary rather than a secondary pigment cell fate. It is only between 30 and 40 h of pupal development that mutant and wild-type discs differ. In pupal discs older than 40 h, no more Elav-positive photoreceptor cells are found in mutant clones. This phenotype is very similar to that caused by the loss of D-APC, a negative regulator of Wingless (Wg) signal transduction. However, genetic analyses reveal that the Gla gain-of-function phenotype can be reverted to wild-type. By generating a P-element-induced revertant of Gla, it has been demonstrated that Gla is allelic to wg. The molecular lesion in Gla indicates that the insertion of a roo retrotransposon leads to ectopic expression of wg during pupal stages. The Gla phenotype is similar to that caused by ectopic expression of Wg driven by the sevenless (sev) enhancer. In both cases Wg exerts its effect, at least in part, by negatively regulating the expression of the Pax2 homolog sparkling (spa). Ectopic expression of wg in sev-wg discs occurs early enough to block the formation of interommatidial bristles by reducing spa expression. In Gla mutants, however, ectopic wg may be expressed too late to interfere with spa expression in the bristle precursor cells, and the sensory organ precursors of interommatidial bristles are formed normally. Ectopic Wg might inhibit a process that normally protects the developing photoreceptor cells from undergoing programmed cell death. Gla represents not only the first dominant allele of wg, but it may also be the first allele ever described for wg (Brunner, 1999).

eyelid antagonizes wingless signaling during Drosophila development and affects patterning of the eye imaginal disc. eyelid was originally isolated as a suppressor of a dominant mutation in the rough gene roughDOMINANT. Eye discs from roDOM third-instar larvae display a so-called "furrow-stop" phenotype, the hallmarks being the unusual presence of mature ommatidia that contain a full complement of photoreceptor cells in the anterior-most ommatidial row, and the loss of dpp expression in the furrow. Surprisingly, an analysis of eye discs from larvae heterozygous for both roDOM and eyelid reveals that the observed increase in eye size is not attributable to relief of the roDOM-induced block to furrow progression in the central region of the disc. Rather, photorecepter differentiation reinitiates at the dorsal and ventral edges of these discs. This produces two ommatidial fields, each preceded by decapentaplegic expressing furrows, which move anteriorly, presumably fusing later along the midline. The mechanism of roDOM suppression by eyelid suggests that eyelid is most critical at the lateral edges of the disc, regions in which wingless has been shown to inhibit precocious differentiation. A reduction of wingless has the opposite effect on roDOM, reducing the eye size and the extent of photoreceptor differentiation along the disc margins. Thus eyelid mutations show opposite effects to those of wingless mutations, suggesting that eyelid may normally function as an antagonist of wg signaling (Treisman, 1997).

Clones of eyelid mutant cells induced in the wing disc also produce pattern alterations suggestive of antagonism to wingless. One effect of clones produced early in development is the transformation of the posterior notum into a partial second wing. These wings have a reversed anterior-posterior polarity; their most clearly differentiated structure is an alula produced consistently at their anterior margin. This transformation is the reverse of that produced by the wingless1 mutation, which transforms the wing into a duplicated notum, and is similar to that produced by overexpressing wingless, decapentaplegic or optomotor-blind in the notum. Clones induced later in wing development are associated with ectopic wing margin bristles. Many or all of these ectopic bristles are not mutant for eld but they are sometimes seen to form adjacent to eld clones. Ectopic bristle formation is restricted to the dorsal surface of the wing within the anterior compartment, and is observed most commonly near the wing margin in tufts of the bristle type appropriate to their postion along the anterior-posterior axis. Ectopic wing margin bristles are also produced in clones mutant for shaggy. However, shaggy clones show neither the non-autonomy nor the positional restrictions observed for eld clones. These results suggest a cell non-autonomous role for eld in wing patterning (Treisman, 1997).

The Drosophila retina is made from hundreds of asymmetric subunit ommatidia arranged in a crystalline-like array, with each unit shaped and oriented in a precise way. One explanation for the precise cellular arrangements and orientations of the ommatidia is that they respond to two axes of polarized information present in the plane of the retinal epithelium. Earlier work has shown that one of these axes lies in the anterior/posterior(A/P) direction and that the polarizing influence is closely associated with the sweep of the Hedgehog-dependent morphogenetic wave. Evidence is presented for a second and orthogonal axis of polarity: this signal can be functionally separated from the A/P axis (see Specification of the eye disc primordium and establishment of dorsal/ventral asymmetry). The polarizing information acting in this equatorial/polar axis (Eq/Pl) is established in at least two steps -- the activity of one signaling molecule functions to establish the graded activity of a second signal. Ectopic Wg expression results in two significant effects. (1) Clones are generated with associated polarity inversions. (2) Although significant changes in retinal polarity are associated with the clones, the distance over which the effect is exerted is restricted to from between 7 to 2 ommatidial rows. Ectopic Wg clones have two distinct features with respect to their polarity effects: (1) the aberrant polarity is asymmetrically distributed in relation to the clone (greater changes in polarity occur in polar positions relative to the center of the clone), and (2) the potency of the Wg-expressing clones to induce polarity reversals show maximial polarity-reversal effects at the equator and minimal effects at the pole (Wehrli, 1998).

Other genes downstream of wingless also appear associated with eye Eq/Pl polarity. The product of the arrow (arr) gene has been placed in the Wingless pathway based on a number of criteria:

To a variable extent, clones of armadillo and dishevelled induce polarity inversions on their equatorial side. The critical observation is that mutations in these recognized transducers of the Wg signal induce non-autonomus effects, consistent with their regulating the activity of a sendary signaling factor. This secondary signal is termed factor-X. Not only do arr, arm and dsh clones specifically affect the equatorial side, they are also more potent in achieving this at the pole than the equator. Thus it is inferred that factor-X activity is graded in the Eq/Pl axis but there is insufficient information to determine whether the activity is high at the equator and low at the poles, or vice-versa (Wehrli, 1998).

Neuronal differentiation in the Drosophila retinal primordium (the eye imaginal disc) begins at the posterior tip of the disc and progresses anteriorly as a wave. The morphogenetic furrow (MF) marks the boundary between undifferentiated anterior cells and differentiating posterior cells. Anterior progression of differentiation is driven by Hedgehog, synthesized by cells located posterior to the MF. hedgehog , which is expressed prior to the start of differentiation along the disc's posterior margin, also plays a crucial role in the initiation of differentiation. Using a temperature-sensitive allele it has been shown that hh is normally required at the posterior margin to maintain the expression of both decapentaplegic (dpp) and the proneural gene atonal. In addition, ectopic differentiation driven by ectopic dpp expression or loss of wingless function requires hh. Consistent with this is the observation that ectopic dpp induces the expression of hh along the anterior margin even in the absence of differentiation. Taken together, these data reveal a novel positive regulatory loop between dpp and hh that is essential for the initiation of differentiation in the eye disc (Borod, 1998).

Dual role for Drosophila epidermal growth factor receptor signaling in early wing disc development: The mechanisms by which wg and vn specify alternate cell fates in the early wing disc, wing, or notum are antagonistic

Cell fate decisions in the early Drosophila wing disc assign cells to compartments (anterior or posterior and dorsal or ventral) and distinguish the future wing from the body wall (notum). Egf receptor signaling stimulated by its ligand, Vein, has a fundamental role in regulating two of these cell fate choices: (1) Vn/EGFR signaling directs cells to become notum by antagonizing wing development and by activating notum-specifying genes; (2) Vn/EGFR signaling directs cells to become part of the dorsal compartment by induction of apterous, the dorsal selector gene, and consequently also controls wing development, which depends on an interaction between dorsal and ventral cells (Wang, 2000).

To determine when Vn/EGFR signaling is required for notum development, the temperature-sensitive alleles, Egfrtsla and vntsWB240 were used. Inactivating Vn/Egfr activity during the second instar (a 24 hr period) causes loss of the notum. The wing develops but shows pattern abnormalities characteristic of vn hypomorphs. Later shifts during the third instar does not cause loss of the notum. This demonstrates that Vn/Egfr activity is required for notum development in the second instar when wg is required to specify the wing. Thus, Vn and Wg appear to have complementary roles and this relationship has been examined by following their expression in mutants (Wang, 2000).

In second instar wild-type wing discs, wg is expressed distally in a wedge of anterior ventral cells and vn is expressed proximally. In vn null mutants, the initiation of wg expression is normal as is expression of its target gene optomotor-blind (omb). In wg mutants, however, there is a dramatic and early expansion of vn expression to include distal cells, presaging the development of these cells as an extra notum. Together these results suggest that Vn has an early role in establishing the notum and that Wg signaling is required to define a distal domain that is reduced in Egfr activity to allow wing development (Wang, 2000).

It is suggested that the mechanisms by which wg and vn specify alternate cell fates in the early wing disc, wing, or notum are antagonistic. This is based on the observation that loss of Wg results in the spread of vn expression and the supposition that the resulting ectopic Egfr activity causes loss of the wing and a double notum phenotype. Further evidence that Vn/Egfr signaling represses wing development comes from the results of misexpressing a constitutive receptor, Egfrlambdatop4.2, in the presumptive wing. In these flies, the wing is reduced to a stump covered with sensilla characteristic of the proximal wing (hinge) region and expression of the wing specific gene vestigial (vg) is repressed. Ectopic notal structures also form from the ventral pleura. The ability of ectopic Egfr signaling to suppress wing development is cell autonomous because clones of cells expressing Egfrlambdatop4.2 lack vg expression. In adult wings these clones produced outgrowths lacking wing characteristics but are otherwise difficult to characterize (Wang, 2000).

Although vn expression expands in wg mutants, no reciprocal spread of wg expression was observed in vn mutants that would have been indicative of a double wing phenotype. However, when Vn/Egfr signaling is inhibited in the notum by expressing a ligand antagonist (Vn::Aos-EGF) under the control of ptc-Gal4, ectopic wings are induced in ~10% of the flies. This result demonstrates that presumptive notal tissue can be transformed to wing by reducing Egfr signaling. However, the transformation occurs only when Egfr signaling is reduced in a subset of cells, rather than all cells in the notum (as in a vn mutant). This may reflect the indirect requirement for Egfr activity to also promote wing development (Wang, 2000).

The loss of notum phenotype is characteristic of vn hypomorphs but in null vn alleles and some Egfr alleles both the wing and notum primordia fail to develop and the wing discs remain tiny. Thus, although ectopic activity of Egfr in the distal disc represses wing development, the pathway is nevertheless normally required for wing development. Using the temperature-sensitive Egfrtsla allele it was found that this requirement is restricted to the period from mid-first to mid-second instar. Key genes involved in wing development that are active at this time include wg and apterous (ap). ap is expressed in dorsal cells and acts as a selector gene to divide the disc into dorsal and ventral compartments. Regulation of Notch ligands by Ap leads to Notch signaling at the DV boundary and the formation of an organizer for wing outgrowth and expression of the wing-specific transcription factor vg (Wang, 2000 and references therein).

Of these two candidates, wg and ap, it seemed unlikely that wg was the key gene affected by Egfr signaling from mid-first to mid-second instar because wg expression is normal in vn mutants at mid-second instar. However, later in the second instar, wg expression normally expands to fill the growing wing pouch and it was noted that in vn mutants, wg expression fails to undergo this expansion. A similar defect in wg expression is seen in ap mutants consistent with Ap function being impaired in vn mutants. Remarkably, ap expression is completely absent in second instar vn mutant discs. Thus, loss of Ap can explain why there is no wing in vn mutants. This is supported by the demonstration that ectopic ap is capable of rescuing wing development in vn mutants (Wang, 2000).

Several additional lines of evidence demonstrate that ap is a cell autonomous target of Vn/Egfr signaling and that this relationship exists only transiently in early wing development: (1) ap expression partially overlaps that of vn in the second instar; (2) ap can be induced ectopically in ventral clones misexpressing an activated form of the receptor, Egfrlambdatop4.2; (3) Egfrtsla mutant clones generated in the first instar show autonomous loss of ap expression, whereas clones generated in the second instar express ap normally. Finally, loss of Egfr activity in whole discs from mid-first to mid-second instar results in complete loss of ap expression, whereas ap is still expressed in discs from larvae given a temperature shift slightly later during the second instar (Wang, 2000).

The results described here suggest that division of the early wing disc into presumptive wing and body wall regions is defined by the action of two secreted signaling molecules, Wg and Vn. wg, a pro-wing gene, is required to repress vn expression, which at high levels antagonizes wing development. Antagonism between Wg and Egfr signaling has also been demonstrated in segmental patterning of the embryo and in development of the head and third instar wing pouch, suggesting such a relationship between these pathways may be a common theme in a number of cell fate choices. Finding that one of the main functions of Wg in early wing specification is to repress Vn/Egfr signaling in the distal region of the early disc raises the question as to whether this is the only role of Wg in wing specification and hence if wing-cell fate can be specified in the absence of both signals. This seems unlikely, because nubbin, an early wing cell marker, is not misexpressed proximally in a vn mutant, where cells would lack both signals (Wang, 2000).

Vn/Egfr signaling promotes development of the notum by maintaining its own activity through transcriptional activation of vn itself, and also promotes expression of ap. Thus, both vn and ap appear to be targets of Egfr signaling, but the domain of ap is clearly wider than that of vn, indicating that ap can be activated at a lower signaling threshold than vn. Vn is a secreted molecule and thus could generate a gradient of Egfr activity. This provides an explanation for how Egfr signaling can regulate both wing and notum development: vn autoregulation and notum development requires high Egfr signaling activity while ap expression and subsequent wing development requires lower signaling activity (Wang, 2000).

Interestingly, vertebrate Egfr and its ligands are expressed in the chick limb bud in a pattern that appears to overlap with the vertebrate ap homolog Lhx2, and these factors are required for limb outgrowth in the chick. In light of the present results it will be important to determine whether Egfr signaling controls Lhx2 expression and thus plays a role in regulating outgrowth of the vertebrate limb. These results may also have implications for the evolution of insect wings. If the control of body wall development by Egfr signaling is ancestral, and comparative analysis of other arthropods will be required to assert this, then one of the first steps towards evolution of wings could have occurred when Egfr signaling assumed control of ap (Wang, 2000).

A polarity field is established early in the development of the Drosophila compound eye

The photoreceptors within the ommatidia of the Drosophila compound eye form a trapezoid. This occurs in two chiral forms in the dorsal and ventral half of the eye. Ommatidia in the dorsal half of the compound eye are oriented with the R3 photoreceptor cell dorsal and anterior, the R7 photoreceptor being ventral. Ommatidia in the ventral half of the eye are inverted. This asymmetry is established during the progression of the morphogenetic furrow as it moves across the epithelium of the eye imaginal disc from posterior to anterior. As the furrow moves it lays down a new column of ommatidial clusters roughly once every 2 hours. However, the ommatidial clusters in one column are not initiated at the same moment, i.e. the first cluster is formed at the center of the furrow (the midline or future equator); subsequent clusters are formed dorsal and ventral to this at about 10-min intervals. This point at the center of the furrow is known as the firing center, an inductive node which transmits information in two directions, i.e. induction of new ommatidial columns towards the anterior and induction of new ommatidial clusters towards the dorsal and ventral poles (Reifegerste, 1997 and references).

Two manipulations were used to induce ectopic ommatidia, in combination with molecular markers for specific positions in the retinal field. Ectopic furrows were generated by shift of winglessl-12 homozygotes to a nonpermissive temperature for 48 hours. Loss of function patched clones were used to induce ectopic furrows, because patched functions as a negative regulator of furrow initiation. Ectopic morphogenetic furrows induced on the eye field margin (or midline) and those induced in the body of the field have different consequences for the establishment of retinal polarity. Ectopic clones on the midline or margin is associated with ectopic expression of early markers of retinal field polarity, while ectopic expression of clones that do not lie on the margin or midline are not associated with such markers. In cases where clones fail to induce ectopic furrows, such clones can re-specifiy polarity field markers if they lie on the margin or midline. Photoreceptor cells in the ectopic ommatidia formed by patched clones produce axons that do not always follow the normal polarity field toward the posterior and the optic stalk. In cases in which a field of ectopic ommatidial clusters is still disconnected from those formed by the endogenous field, the ectopic clusters do not find a path to the optic stalk, but converge on the center of their local field. This phenomenon may be similar to the development of axon tracts in the insect central nervous system and is consistent with a homophilic axon guidance model (Reifegerste, 1997).

An early equatorial model for retinal polarity is proposed. In this model, early events establish the dorsal/ventral polarity of the retinal field and establish the midline/equator; only later does the furrow initiate and then the firing center follows the midline, but does not form it. This idea is derived from the observation that markers of polarity are expressed in specific parts of the retinal field before furrow initiation. Thus events that initiate furrow movement on the margin or the midline re-specify the field markers, while those that lie off the margin or the midline do not. Evidence for a preexisting field of positional information comes from the characterization of the homeoprotein mirror, which seems to be involved in the establishment of retinal polarity. The gene four-jointed shows a graded expression in equatorial-polar direction along the equator in third instar eye imaginal discs. Four jointed is a putative cell surface or secreted protein. Another candidate for an equatorial signal is Wingless itself. Wingless could act early to signals from the margins inwards. A second signal from the midline could be induced by early Wingless. Mosaic clones for frizzled affect retinal polarity; these have a domineering non-autonomy on adjacent wild type tissue. Proteins similar to Frizzled have been shown to act as Wnt receptors (Reifegerste, 1997 and references).

The Drosophila Jak kinase Hopscotch is required for multiple developmental processes in the eye

Jak kinases are critical signaling components in hematopoiesis. While a large number of studies have been conducted on the roles of Jak kinases in the hematopoietic cells, much less is known about the requirements for these tyrosine kinases in other tissues. Loss of function mutations in the Drosophila Jak kinase Hopscotch (Hop) were used to determine the role of Hop in eye development. Hop is required for cell proliferation/survival in the eye imaginal disc, for the differentiation of photoreceptor cells, and for the establishment of the equator and of ommatidial polarity. These results indicate that hop activity is required for multiple developmental processes in the eye, both cell-autonomously and nonautonomously (Luo, 1999).

Mutations in both wingless signaling components and hop affect equator formation nonautonomously, suggesting that a secondary diffusible signal exists downstream of them. However, their phenotypes are different in two ways: (1) mutants in wingless pathway components affect polarity on the equatorial side of the clone, whereas in hop mutant clones the polarity is reversed on the polar side, and (2) an ectopic equator forms in the center of a wingless pathway mutant clone, whereas such an ectopic equator forms outside a hop clone. The wg-signaling data suggest that (in addition to its indirect role on fng expression and thus Notch activation) Wg signaling controls a diffusible factor as a secondary signal that is either up- or down-regulated (depending on whether it is a positive or negative factor. The nonautonomy of the hop mutant clones also suggests that a secondary diffusible signal acts downstream of Hop. However, it might act in the opposite direction from the one regulated by Wg due to the opposite influence on polarity by hop- and wg-signaling components, respectively. The full understanding of the nature of the Wg- and Hop-associated phenotypes, and of their potential interactions and secondary signals activated, will only be possible when the presumptive secondary signals are identified (Luo, 1999)

Regulation of dally, an integral membrane proteoglycan, and its function during adult sensory organ formation of Drosophila

In Drosophila, imaginal wing discs, Wg and Dpp, play important roles in the development of sensory organs. These secreted growth factors govern the positions of sensory bristles by regulating the expression of achaete-scute (ac-sc), genes affecting neuronal precursor cell identity. Earlier studies have shown that Dally, an integral membrane, heparan sulfate-modified proteoglycan, affects both Wg and Dpp signaling in a tissue-specific manner. dally is required for the development of specific chemosensory and mechanosensory organs in the wing and notum. dally enhancer trap is expressed at the anteroposterior and dorsoventral boundaries of the wing pouch, under the control of hh and wg, respectively. dally affects the specification of proneural clusters for dally-sensitive bristles and shows genetic interactions with either wg or dpp signaling components for distinct sensory bristles. These findings suggest that dally can differentially regulate Wg- or Dpp-directed patterning during sensory organ assembly. For pSA, a bristle on the lateral notum, dally shows genetic interactions with iroquois complex (IRO-C), a gene complex affecting ac-sc expression. Consistent with this interaction, dally mutants show markedly reduced expression of an iro::lacZ reporter. These findings establish dally as an important regulator of sensory organ formation via Wg- and Dpp-mediated specification of proneural clusters (Fujise, 2001).

dally cooperates with Wg to form the wing margin structures. Ac expression is severely decreased in dally mutants, supporting the idea that dally serves as a component of the Wg receptor complex to induce AS-C expression at the prospective wing margin. Taken together, these observations indicate that dally is a target gene of Wg signaling pathway, and at the same time, it mediates the same signaling, suggesting that dally is involved in a positive feedback loop of Wg signaling at the D/V boundary of the wing pouch. Frizzled-2, the Wg receptor, is down-regulated by Wg signaling at the D/V boundary. Dally, a putative Wg coreceptor, may also participate in the feedback circuits of Wg signaling, as has been suggested for Fz2 (Fujise, 2001).

Wg and Dpp have been shown to affect prepatterning of sensory organs by governing the expression of proneural genes, such as ac-sc. dally has been shown to affect the signaling levels of either Wg or Dpp. Therefore, an examination was made to determine whether dally affects sensory organ formation via either Wg or Dpp signaling pathways. Genetic experiments provided evidence that, in the prospective notum region of the wing disc, dally selectively influences Wg signaling to form the pPA bristle and Dpp signaling to form the pSA and DC bristles. It is particularly intriguing that, during development of DC macrochaetae, dally genetically interacts with only Dpp signaling, while the formation of these bristles requires both Wg and Dpp activities. It has been indicated that the A/P coordinates of the DC cluster are limited by Dpp signaling. In dally homozygous wing discs, the DC cluster is apparently shorter in the A/P coordinates compared with wild-type discs, suggesting that dally regulates Dpp signaling activity to limit the A/P length of the DC cluster. What are the mechanisms that can account for the selective interactions of dally and specific growth factor signaling? One obvious interpretation of genetic experiments on DC macrochaetae is that differences in dose effects between dpp and wg are responsible for the apparent specificity. It is also possible that the ligand-specificity of Dally is controlled at the cellular level through modification of heparan sulfate structures (Fujise, 2001).

The tissue polarity gene nemo carries out multiple roles in patterning during Drosophila development

The murine Nemo homolog Nlk has been implicated in regulating Wnt signaling by repressing the Arm/ß-Catenin-TCF complex, a component of Wingless signaling. Whether such an interaction occurs in flies during wing vein formation was examined. The extra vein phenotypes observed in nmo mutant wings are very similar to those that have been previously described as resulting from overexpression of Armadillo and both vertebrate ß-catenin and plakoglobin in the wing. In addition, ectopic veins are produced as a result of ectopic wg and dsh expression. Constitutively active Armadillo (UAS-Arms10) expressed using the 1348-Gal4 driver leads to the formation of moderate ectopic veins emanating from the PCV, in addition to more severe ectopic veins along LII. These are both regions of the wing that are sensitive to nmo mutations and where similar ectopic veins are observed in nmoadk. The phenotypes seen with overexpression of wg and arm are consistent with the theory that Nemo is a negative regulator of Wingless signaling since loss of nemo mimics extra veins seen with overexpression of arm and wg (Verheyen, 2001).

Overexpression of both wildtype mouse Lef-1 (a TCF homolog) and a constitutive repressor form of dTCF (Pangolin) results in dominant negative phenotypes. The constitutive repressor form of dTCF (UAS-dTCFDeltaN) is unable to bind Arm and represses wg-dependent gene expression. These findings suggest that expression of wildtype dTCF (UAS-dTCFwt) somehow interferes with a wingless-targeted transcription factor in a dominant negative way. Consistent with this, it is found that ectopic expression of UAS-dTCFwt using vestigial-Gal4 results in defects in the posterior wing margin, a phenotype seen with loss of wg signaling. Ectopic expression of the UAS-dTCFDeltaN using the 1348-Gal4 driver is lethal. However, homozygosity for nmoadk is able to rescue the lethality and the flies that emerged had reduced ectopic wing veins. This finding can be interpreted by taking into account the dual roles TCF plays in the nucleus. In nmoadk mutants, the negative regulation of endogenous dTCF may be reduced, leading to more wg-dependent signaling and the induction of extra veins similar to those seen with constitutive Arm expression. Expression of UAS-dTCFDeltaN in the nmoadk background most likely interferes with the de-repressed endogenous dTCF and block the induction of extra veins seen in nmoadk (Verheyen, 2001).

Genetic requirements of vestigial in the regulation of Drosophila wing development

The gene vestigial has been proposed to act as a master gene because of its supposed capacity to initiate and drive wing development. The ectopic expression of vestigial only induces ectopic outgrowths with wing cuticular differentiation and wing blade gene expression patterns in specific developmental and genetic contexts. In the process of transformation, wingless seems to be an essential but insufficient co-factor of vestigial. vestigial ectopic expression alone or vestigial plus wingless co-expression in clones differentiate 'mixed' cuticular patterns (they contain wing blade trichomes and chaetae characteristic of the endogenous surrounding tissue) and express wing blade genes only in patches of cells within the clones. In addition, these clones, in the wing imaginal disc, may cause autonomous as well as non-autonomous cuticular transformations and wing blade gene expression patterns. These non-autonomous effects in surrounding cells result from recruitment or 'inductive assimilation' of vestigial or wingless-vestigial overexpressing cells (Baena-López, 2003).

The notion of 'master gene', as applied to the gene eyeless, corresponds to a gene that by itself would trigger a developmental program that is independent of the tissue where it is expressed. Although this definition has been applied to vg, the present results indicate otherwise. The ectopic expression of vg elicits certain characteristics of 'wing blade' development but is not sufficient for a complete transformation. The effect of vg depends on the time and genetic context of the tissue where it is overexpressed. These results reveal a strong dependence of vg on wg to initiate a wing blade developmental pathway. Wg by itself does not lead to tissue transformations. This cooperative effect between wg and vg remains insufficient in all tissues analyzed, suggesting the existence of additional genes necessary to initiate and drive wing development. The molecular mechanisms that underlie the interaction between wg pathway and vg are not known. However, the co-expression of vg with a construct of armadillo (arm) (transcriptional effector of wg pathway) using vg-G4 fails to promote the transformation of eye tissue. This result suggests that the interaction of wg and vg takes place upstream of arm and, therefore, outside of the cell nucleus. Whereas vg requires high levels of wg expression to initiate wing development, the clones of vg overexpression contain in later stages, low or null levels of wg expression. Moreover, wg-vg co-overexpression clones can also show low levels of wg, even when wg is also mobilized in G4 territories or in Flip-out clones. These results suggest that vg may indirectly reduce wg expression once wing development is already initiated, and may explain why the transformed tissue in vg clones does not contain wing margin cuticular elements. The late repression of wg seems to be important to specify territories of the wing blade depending on vg expression outside of the wing margin; if high levels of Wg are maintained, all cells differentiate into wing margin chaetae. It is concluded that wg and vg activities together specify wing margin territories, but vg alone specifies the remaining part of the wing blade (Baena-López, 2003).

The ectopic expression of vg or wg-vg in clones may cause outgrowths with wing histotypic characteristics or patterning perturbations in the notum, leg or eyes. The transformed tissues show 'mixed' phenotypes or 'mosaic' territories where, in a 'salt and pepper' distribution, wing blade trichomes co-exist with notum or leg chaetae. Adult cuticular 'mixed' phenotypes are correlated with the ectopic expression of wing blade genes in particular combinations. However, expression of wing blade genes is detected only in some compact groups of cells within the clones. These results indicate that either vg or wg-vg are insufficient by themselves to displace all endogenous signals of identity, or that reciprocal non-autonomous influences between clonal cells and surrounding cells exist, reducing the expression of wing blade genes to groups of cells within clones. The change of wing blade genes expression in compact groups of cells in the disc and 'mixed' (salt and pepper) cuticular phenotypes in the adult could result from cell interactions during patterning and cell rearrangements in pupal stages (Baena-López, 2003).

Transformations induced by overexpression of vg or wg-vg in clones and G4 territories are, as a rule, cell autonomous, except in the wing hinge, notum and corresponding tissues in the haltere. In the wing hinge the cells of the outgrowths outside the vg clones differentiate into wing blade territories and show gene expression patterns characteristic of the wing blade cells located between the proximal vg expression and the internal ring of wg in the wild-type disc. This suggests that the non-autonomous effects in vg clones could reproduce the wild-type intercalary growth induced by the confrontation of cells expressing proximal genes with distal genes. In the notum, vg clones located simultaneously in territories expressing and not expressing ap, and initiated in the wg expression domain, may non-autonomously recruit surrounding cells to express characteristic wing blade genes at long cell distances, as wg-vg clones do. Thus, vg (together with wg expression) is necessary to induce and extend the transformation over long distances outside the clones. In contrast to vg or wg-vg clones, wg clones do not show non-autonomous transformation phenotypes and expression of wing blade genes at long distances. The issue of whether the recruitment process is caused by Wg diffusion, or whether it results from intercalary growth induced by the confrontation between cells expressing proximal genes (genes of the notum) and cells expressing distal genes (wing blade genes), remains unresolved (Baena-López, 2003).

The expression of selector genes like Ubx and en is not modified by overexpression of vg or wg-vg, but is inherited and maintained. However, the expression of the selector gene ap can be modified or inherited in some tissues, such as the legs, to give DV identity (Baena-López, 2003).

The comparative analysis of vg with other morphogenetic genes suggests that vg acts as Dll, pnr or iro, rather than as a 'master' or 'selector of tissue' gene: vg is simply a component of the genetic combination that is necessary to initiate and drive wing blade development where vg is normally expressed. Interestingly, the function of vg, in addition to conferring territorial identity, may also non-autonomously recruit surrounding cells ('inductive assimilation'), changing their specific cuticular and gene expression patterns. This is related to its function as a local organizer of growth when it is expressed among cells with different positional or regional fates. Later in development, vg, in combination with other genes, activates an inventory of downstream wing genes that specify more discrete territories within the wing blade such as veins, interveins and sensory elements (Baena-López, 2003).

Wingless promotes cell survival but constrains growth during Drosophila wing development

During animal development, organs grow to a fixed size and shape. Organ development typically begins with a rapid growth phase followed by a gradual decline in growth rate as the organ matures, but the regulation of either stage of growth remains unclear. The Wnt/Wingless (Wg) proteins are critical for patterning most animal organs, have diverse effects on development and have been proposed to promote organ growth. This study reports that contrary to this view, Wg activity actually constrains wing growth during Drosophila wing development. In addition, Wg is required for wing cell survival, particularly during the rapid growth phase of wing development. It is proposed that the cell-survival- and growth-constraining activities of Wg function to sculpt and delimit final wing size as part of its overall patterning program (Johnston, 2003).

Hedgehog promotes Bowl protein accumulation by promoting drm expression, while Wingless antagonizes Hedgehog function and Bowl accumulation by repressing drm expression

The operation of the Drm/Lines/Bowl regulatory pathway was examined in the context of the epidermal organizer. Across the dorsal embryonic epidermis, Hedgehog and Wingless are the key pattern-organizing signals. Hedgehog specifies cell fate in half the PS (the 1°-3° cell fates), while Wingless specifies the remaining cell fate (the 4° cell fate) in the complementary half. To investigate whether Hedgehog and Wingless engage the Drm/Lines/Bowl regulatory pathway, drm gene expression and Bowl protein accumulation were examined under conditions of loss or excess of Hedgehog or Wingless signaling. Expression of drm was found to be decreased in hedgehog mutants, and expanded posteriorly in embryos expressing the secreted form of Hedgehog in Engrailed/Hedgehog-expressing cells. Two points are noteworthy here: (1) while Hedgehog can directly control drm expression posterior to the Hedgehog domain, control within the Hedgehog domain is likely indirect since these cells cannot themselves respond to Hedgehog signaling; (2) the fact that excess Hedgehog does not induce drm expression in anterior cells suggests that Wingless signaling represses drm expression in this region. Consistent with this prospect, it was found that drm expression is ectopically activated in wingless mutants and repressed upon ectopic activation of the Wingless pathway. It was also found that changes in drm expression due to manipulations of Hedgehog and Wingless signaling largely led to the expected changes in Bowl protein accumulation. For instance, broadened drm expression caused by excess Hedgehog leads to a broadened Bowl domain, while the ectopic stripe of drm expression in wingless mutants also leads to increased Bowl accumulation, although Bowl accumulates rather more broadly than the narrow drm stripe would suggest. These changes in Bowl accumulation correlate nicely with the patterning changes observed with inactivation or activation of Hedgehog or Wingless signaling. It is concluded that the asymmetric response of drm to Hedgehog underlies the pattern of epidermal cell differentiation since drm promotes the accumulation of Bowl in drm-expressing cells and consequent cellular responses elicited by Bowl. Note that Bowl accumulates in two rows of cells but apparently is required for patterning across a broader region. This observation implies that Bowl controls expression of a new signal that further elaborates epidermal pattern (Hatini, 2005).

ecruitment of cells into the Drosophila wing primordium by a feed-forward circuit of vestigial autoregulation

The Drosophila wing primordium is defined by expression of the selector gene vestigial (vg) in a discrete subpopulation of cells within the wing imaginal disc. Following the early segregation of the disc into dorsal (D) and ventral (V) compartments, vg expression is governed by signals generated along the boundary between the two compartments. Short-range DSL (Delta/Serrate/LAG-2)-Notch signaling between D and V cells drives vg expression in 'border' cells that flank the boundary. It also induces these same cells to secrete the long-range morphogen Wingless (Wg), which drives vg expression in surrounding cells up to 25-30 cell diameters away. Wg signaling is not sufficient to activate vg expression away from the D-V boundary. Instead, Wg must act in combination with a short-range signal produced by cells that already express vg. Evidence that this vg-dependent, vg-inducing signal feeds forward from one cell to the next to entrain surrounding cells to join the growing wing primordium in response to Wg. It is proposed that Wg promotes the expansion of the wing primordium following the D-V segregation by fueling this non-autonomous autoregulatory mechanism (Zecca, 2007a; full text of article)

Following the D-V segregation, local DSL-Notch signaling across the compartment boundary induces the differentiation of specialized border cells that express vg, secrete Wg, and organize a dramatic ~200-fold expansion of the wing primordium. In ap0 wing discs, D-V segregation fails to occur, border cells are not specified, and the early expression of vg that initially defined the wing primordium fades away. This mutant condition was used to explore how vg and wg activity in border cells controls wing growth by asking what happens when the missing border cells were replaced with cells that ectopically express Wg, Vg or both (Zecca, 2007a).

The main finding of this study is that Wg is not sufficient to sustain or induce vg expression in ap0 discs, even when the morphogen is overexpressed, continuously, in all cells. Instead, Wg can only drive vg expression in these discs when the responding cells are near or next to cells that express exogenous Vg. The clearest demonstration of this is the experiment in which two types of clones were generated in the same ap0 disc: clones that express Nrt-Wg, a membrane tethered immobile form of Wg, and clones that express moderate levels of exogenous Vg. Neither type of clone, alone, can restore normal expression of the endogenous vg gene. However, ectopic Vg-expressing clones can induce neighboring Nrt-Wg-expressing clones to express vg, provided that they abut. Moreover, this vg expression can spread through the Nrt-Wg-expressing clone and extend to adjacent cells outside the clone (Zecca, 2007a).

These results indicate that vg-expressing cells send a short-range, possibly contact-dependent signal that is required to entrain neighboring cells to express vg in response to Wg. Furthermore, they indicate that once the responding cells express vg, they can in turn entrain their neighbors in the same way, propagating the recruitment of additional cells into the wing primordium. These findings establish the existence of a Wg-dependent feed-forward circuit of vg autoregulation and suggest that D-V border cells normally organize wing growth by providing Wg, as well as the initial Vg-dependent entraining signal that triggers reiteration of this autoregulatory circuit from one cell to the next (see feed-forward circuit of Wg-dependent vg autoregulation in Drosophila). Thus, feed-forward regulation in this context has a spatial component, mediating the expansion (in mass and cell number) of a developing primordium by a process of recruitment (Zecca, 2007a).

These results are concordant with previous reports that Wg signaling cannot drive vg expression in the wing imaginal disc in the absence of border cells, and that co-overexpression of Wg and Vg can synergize to drive vg expression in surrounding cells. However, the current findings advance these results in three significant ways. First, it was shown that vg-expressing cells provide a discrete second signal, required together with Wg, to induce vg expression in surrounding cells. Second, it was demonstrated that production of this signal can propagate from one cell to the next, establishing a feed-forward autoregulatory mechanism fueled by morphogen. Third, it was shown that physiologically normal levels of wg and vg activity are sufficient to initiate and propagate this feed-forward mechanism, establishing that it is a natural process and not an overexpression artifact (Zecca, 2007a).

The capacity of Wg to drive recruitment of new cells into the wing primordium by fueling vg feed-forward autoregulation provides one mechanism for promoting wing growth. However, it appears to operate within the context of other mechanisms for promoting wing growth, as well as for limiting where and when such growth occurs. At least three additional mechanisms for promoting wing growth, all dependent on Wg, an be distinguished. First, in addition to recruiting new cells into the wing primordium, Wg acts continuously to retain cells that were previously recruited: wing cells in which Wg transduction is abrogated rapidly lose vg expression and either die, or sort out. It is suggested that retention, like recruitment, depends on the same Wg-dependent vg autoregulatory circuit. Specifically, it is posited that the feed-forward signal is required both to induce vg expression in cells about to enter the primordiium, as well as to maintain vg expression in cells after they enter (Zecca, 2007a).

Second, independent of its role in fueling vg autoregulation, Wg also appears necessary for the survival and proliferation of vg-expressing wing cells. It is possible to bypass the requirement for Wg-dependent vg autoregulation by using a Tubalpha1>vg transgene to express exogenous Vg: nevertheless, such 'rescued' Tubalpha1>vg wing cells still require Wg input to survive, grow and proliferate (Zecca, 2007a).

Third, cells are normally recruited into the vg-expressing population from a surrounding population defined by detectable expression of rn but not vg. Accordingly, the 'rn-only' population must proliferate in conjunction with the growth of the wing primordium; otherwise, it would be depleted, limiting further recruitment and compromising the development of more proximal structures. In support, it was found that the rescue of the wing primordium by Wg-dependent vg autoregulation is associated with the rescue and expansion of the surrounding population of rn-only cells. Hence, once cells are recruited into the wing primordium in response to Wg, they may send an additional signal to sustain the source population of rn-only cells from which additional wing cells will be recruited (Zecca, 2007a).

Conversely, at least three mechanisms can be distinguised that appear to constrain operation of the feed-forward circuit, limiting expansion of the wing primordium in space and time. First, is the early segregation of the wing imaginal disc into distinct distal (pre-blade) and proximal (pre-hinge/notum) compartments, only one of which, the pre-blade, is competent to engage the feed-forward autoregulatory circuit. This event, which occurs before D-V compartmental segregation, appears to be governed by an early burst of Wg signaling that selectively and heritably represses tsh expression in the founder cells of the putative pre-blade (tshOFF) compartment. Although Wg-dependent vg autoregulation normally appears to operate only within the resulting pre-blade (tshOFF) compartment (which includes the rn-only domain, as well as the presumptive wing pouch), this limit can be exceeded if cells are exposed to ectopic Wg signal before they would otherwise segregate into the pre-hinge/notum (tshON) compartment. It is suggested that this ectopic Wg activity inappropriately blocks tsh activity in the prospective pre-hinge/notum, creating an ectopic pre-blade compartment in which feed-forward regulation can occur (Zecca, 2007a).

Second, is the availability of Dpp secreted by A compartment cells along the A-P compartment boundary. Dpp, like Wg, is essential for vg expression and wing growth. Hence, operation of the feed-forward mechanism might depend on the combined inputs of Wg and Dpp, centering the expanding domain of Wg-dependent vg expression on the intersection between the D-V and A-P compartment boundaries. In agreement, evidence for Wg-dependent feed-forward propagation is observed only in cells located within ~25 cell diameters of the A-P boundary, the expected range of Dpp emanating from A cells along the boundary (Zecca, 2007a).

Third, operation of the vg feed-forward circuit might be temporally constrained. It is striking that vg is initially expressed in ap-null discs up until the time the D-V compartmental segregation would normally occur; yet, flooding such discs with exogenous Wg signal is not sufficient to sustain and propagate this early vg expression. By contrast, clones of Tubalpha1-vg cells generated in these same discs are effective in triggering the propagation of vg expression in surrounding cells, suggesting that cells within the 'pre-blade' become competent to operate the feed-forward autoregulatory circuit only after the time at which the D-V segregation normally occurs, concomitant with the differentiation of wg- and vg-expressing border cells (Zecca, 2007a).

Thus, it is proposed that following the D-V segregation, Wg drives wing growth by at least four distinct outputs: first, by recruiting new cells into the wing primordium; second, by maintaining the recruited cells and their descendents within the primordium; third, by sustaining the survival and proliferative growth of cells defined as 'wing' by the selector activity of Vg; and finally, by acting through the agency of newly recruited wing cells to induce the expansion of the surrounding population of rn-only cells from which additional wing cells will be recruited. Counterbalancing these effects would be a requirement for heritable repression of tsh, availability of Dpp, and transition to a discrete phase of wing disc development during which the feed-forward circuit can operate. Within these constraints, the size of the wing primordium at any point following the D-V segregation would reflect the increasing range of Wg emanating from the D-V border cells via its capacity to propagate and sustain the vg autoregulatory circuit and, separately, its capacity to promote the proliferative growth of vg- and rn-only-expressing cells (Zecca, 2007a).

Establishment of global patterns of planar polarity during growth of the Drosophila wing epithelium

Epithelial tissues develop planar polarity that is reflected in the global alignment of hairs and cilia with respect to the tissue axes. The planar cell polarity (PCP) proteins form asymmetric and polarized domains across epithelial junctions that are aligned locally between cells and orient these external structures. Although feedback mechanisms can polarize PCP proteins intracellularly and locally align polarity between cells, how global PCP patterns are specified is not understood. It has been proposed that the graded distribution of a biasing factor could guide long-range PCP. However, epithelial morphogenesis has been identified as a mechanism that can reorganize global PCP patterns; in the Drosophila pupal wing, oriented cell divisions and rearrangements reorient PCP from a margin-oriented pattern to one that points distally. This study used quantitative image analysis to study how PCP patterns first emerge in the wing. PCP appears during larval growth and is spatially oriented through the activities of three organizer regions that control disc growth and patterning. Flattening morphogen gradients emanating from these regions does not reduce intracellular polarity but distorts growth and alters specific features of the PCP pattern. Thus, PCP may be guided by morphogenesis rather than morphogen gradients (Sagner, 2012).

To study the emergence of polarity in the wing disc, the subcellular distribution of the PCP proteins Flamingo (Fmi) and Prickle (Pk) were quantified. Planar cell polarity (PCP) nematics were calculated based on Fmi staining and PCP vectors based on the perimeter intensity of EGFP::Pk clones. At 72 hr after egg laying (hAEL), the wing pouch has just been specified and is small. EGFP::Pk localizes to punctate structures at the cell cortex that are asymmetrically distributed in some cells, but PCP vectors exhibit no long-range alignment. By 96 hAEL, PCP vector magnitude increases and a global pattern emerges. Later, PCP vector magnitude increases further and the same global polarity pattern is clearly apparent. It is oriented with respect to three signaling centers: the dorsal-ventral (DV) boundary (where Wingless [Wg] and Notch signaling occur), the anterior-posterior (AP) compartment boundary (where Hedgehog [Hh] and Decapentaplegic [Dpp] signaling occur), and with respect to the hinge fold (where levels of the atypical Cadherin Dachsous [Ds] change sharply) (Sagner, 2012).

PCP vectors in the wing pouch near the hinge fold point away from it toward the center of the pouch. Within the Wg expression domain at the DV boundary, PCP vectors parallel the DV boundary and point toward the AP boundary. Just outside this domain, PCP nematics and vectors turn sharply to point toward the DV boundary in central regions of the wing pouch. However, where the DV boundary intersects the hinge-pouch interface, they remain parallel to the DV boundary over larger distances such that PCP vectors orient away from the hinge around the entire perimeter of the wing pouch (Sagner, 2012).

The AP boundary is associated with sharp reorientations of PCP. First, PCP vectors that parallel the DV boundary point toward the AP boundary in both anterior and posterior compartments. Second, although PCP vectors in the central wing pouch are generally orthogonal to the DV boundary, they deflect toward the AP boundary where Hh signaling is most active (as defined by upregulation of the Hh receptor Patched [Ptc]). On either side of this region, PCP vectors turn sharply to realign parallel to the AP boundary. Third, PCP vectors in the hinge point away from the AP boundary and align parallel to the hinge fold (Sagner, 2012).

The atypical Cadherins Fat (Ft) and Ds limit disc growth and orient growth perpendicular to the hinge. Their loss perturbs the PCP pattern in pupal wings and alters hair polarity. To investigate whether they influence the larval pattern, PCP was was quantified in ft and ds mutant discs. The PCP pattern is similar to wild-type (WT) in the central wing pouch but altered in proximal regions close to the hinge fold. Polarity vectors deviate from their normal orientation (away from the hinge fold) in many regions of the proximal wing pouch. This is especially clear near the intersection of the DV boundary with the hinge - here, PCP vectors orient toward the DV boundary rather than away from the hinge. Furthermore, near the AP boundary, vectors form a reproducible point defect, with vectors pointing away from the defect center (Sagner, 2012).

After pupariation, morphogenesis reshapes the wing disc, apposing its dorsal and ventral surfaces such that the DV boundary defines the margin of the wing blade. During reshaping the PCP pattern evolves, but specific local features are retained through pupal development. Consistent with this, hair polarity in ds adult wings proximal wing near the anterior wing margin orient toward the margin rather than away from the hinge. Near the AP boundary, hairs form swirling patterns. Thus, Ft and Ds are required during larval growth to ensure that PCP vectors in the proximal wing orient away from the hinge (Sagner, 2012).

Notch and Wg signaling at the DV boundary organize growth and patterning in the developing wing. These pathways maintain each other via a positive feedback loop; Notch induces transcription of Wg at the DV interface, and Wg signaling upregulates expression of the Notch ligands Delta (Dl) and Serrate (Ser) adjacent to the Wg expression domain, further activating Notch signaling at the DV boundary. To study how the DV boundary organizer affects PCP, Ser was ectopically expressed along the AP boundary with ptc-Gal4 (ptc > Ser). In the ventral compartment, Ser induces two adjacent stripes of Wg expression, which then upregulate Dl expression in flanking regions (dorsally, Fringe prevents Notch activation by Ser. The posterior Wg and Dl stripes are distinct, but the anterior stripes are broader due to the graded activity of ptc-Gal4. In these discs, the ventral compartment overgrows along the AP boundary, parallel to the ectopic 'organizers'. PCP nematics and vectors near the posterior Wg/Dl stripes are organized similarly to those flanking the normal DV boundary, running parallel to the stripe and turning sharply outside this region to orient toward the ectopic organizer). PCP nematics anterior to the ectopic Ser stripe run parallel to it over larger distances before turning sharply, consistent with the broader Wg/Dl expression in this region. In resulting adult wings, hairs orient toward the ectopic wing margin that forms along the AP boundary. Ectopically expressing Wg along the AP boundary also generates an ectopic organizer that reorients growth and PCP (Sagner, 2012).

To ask how loss of the DV boundary organizer affected PCP, a temperature-sensitive allele of wg was used that blocks Wg secretion (wgTS), or wings were populated with wg null mutant clones. Loss of Wg signaling severs the feedback loop with Notch such that both decay. PCP nematics were quantified in wgTS discs shifted to the restrictive temperature shortly after the second to third-instar transition (earlier, Wg is required to specify the wing pouch). wgTS discs have smaller wing pouches than WT and are missing a large fraction of the central region of the pouch where polarity orients perpendicular to the DV boundary. Polarity still orients away from the hinge, thus the PCP pattern in wgTS discs appears more radial (i.e., oriented toward the center of the wing pouch). Analogously, adult wings populated by wg null clones are missing those regions of the distal wing blade where hairs normally point perpendicular to the wing margin. The remaining proximal tissue is normally polarized except at its distal edges. Here, polarity deflects from the proximal-distal axis to parallel the edge of the wing. Normally, hair polarity in the wing blade parallels the margin only in proximal regions, where Ft/Ds influences polarity. Thus, the DV organizer is needed to orient PCP in distal regions perpendicular to the margin. Ft/Ds is required for a complementary subset of the PCP pattern in the proximal wing. Their influences largely reinforce each other (i.e., away from the hinge and toward the DV boundary or wing margin) except where the hinge and wing margin intersect. Here, loss of one signaling system expands the influence of the other. Wg is distributed in a graded fashion and is a ligand for Frizzled (Fz). Thus, it could bias the PCP pattern directly, e.g., by asymmetrically inhibiting interactions between Fz, Strabismus (Stbm), and Fmi or causing Fz internalization. If so, uniform Wg overexpression should prevent intracellular polarization or reduce cortical localization of PCP proteins. To investigate this, Wg was overexpressed uniformly (C765 > wg::HA). Uniform Wg expression elongates the wing pouch parallel to the AP boundary. It broadens the pattern of Dl expression, such that sharp Dl stripes at the DV boundary are lost, but Dl expression remains excluded from the Hh signaling domain anterior to the AP boundary. Fmi and EGFP::Pk polarize robustly in these discs; thus, the Wg gradient does not act directly on PCP proteins to induce or orient polarity. However, the pattern of PCP vectors and nematics is altered. PCP points away from the hinge (rather than perpendicular to the DV boundary) over larger distances compared to WT and then turns sharply to face theDV boundary in the middle of the wing pouch. Because specific alterations in the PCP pattern are induced by uniform Wg overexpression, Wg protein distribution does not directly specify the new PCP pattern (Sagner, 2012).

To identify signals that influence the PCP pattern near the AP boundary, the effects of uniform high-level expression of Dpp and Hh, two morphogens that form graded distributions near the AP boundary, were examined. Uniform Dpp expression does not influence the magnitude of PCP or the range over which PCP deflects toward the AP boundary. Interestingly, uniform Hh expression dramatically increases the range over which PCP deflects toward the AP boundary, suggesting that Hh is important for this aspect of the pattern. However it clearly indicates that PCP vectors are not oriented directly by the graded distribution of Hh or by the graded activity of Hh signaling, because both are uniformly high in the anterior compartment of Hh overexpressing discs. Whether the apposition of cells with very different levels of Hh signaling might produce sharp bends in the PCP pattern was therefore considered. In WT discs, Hh signaling levels change at two interfaces: one along the AP boundary and one along a parallel line outside the region of highest Hh signaling where Ptc is upregulated. PCP vectors orient parallel to the AP boundary in the cells posterior to it, deflect toward the boundary anteriorly, and then reorient sharply outside of this region to align parallel to the AP boundary. Discs uniformly overexpressing Hh have only one signaling discontinuity (at the AP boundary), because Hh signaling is high throughout the anterior compartment. This could explain why PCP in these discs remains deflected toward the AP boundary over longer distances (Sagner, 2012).

To test this, clones mutant for the Hh receptor Ptc, which constitutively activate signaling in the absence of ligand, were generated. Quantifying PCP nematics in these discs reveals reproducible patterns of polarity reorientation at interfaces between WT and ptc- tissue. In WT tissue adjacent to ptc- clones, PCP aligns parallel to the clone interface. Due to the typical clone shape, this orientation is often consistent with the normal PCP pattern. However, PCP also aligns parallel to ptc- clones in regions where this is not so. Thus, ptc- clones exert a dominant effect on adjacent WT tissue. In contrast, on the mutant side of the clone interface, polarity tends to orient perpendicular to the interface. Thus, apposition of high and low levels of Hh signaling causes a sharp bend in the PCP pattern. Corresponding polarity reorientation by ptc- clones is also seen in adult wing. Thus, Hh signaling has two effects in WT discs: within the Hh signaling domain, it deflects PCP toward the AP boundary, and just outside the Hh signaling domain, it orients PCP parallel to the AP boundary. In this region, the tendency for polarity to align parallel to Hh signaling interfaces is consistent with the orientation of polarity toward the DV boundary and away from the hinge. Thus, these three polarity cues reinforce each other throughout much of the wing pouch, making the global PCP pattern robust (Sagner, 2012).

Simulations have highlighted the difficulty of establishing long-range polarity alignment in a large field of cells from an initially disordered arrangement. The pattern typically becomes trapped in local energy minima, forming swirling defects. Introducing a small bias in each cell removes such defects - this has been an attractive argument for the involvement of large - scale gradients in orienting PCP. The graded distribution of Ds along the proximal-distal axis (orthogonal to the hinge-pouch interface) suggested a plausible candidate for such a signal. Strikingly, the Ds expression gradient gives rise to intracellular polarization of both Ft and Ds, and the recruitment of the atypical myosin Dachs to the distal side of each cell. Nevertheless, most of the PCP defects in ft mutants can be rescued by uniform overexpression of a truncated Ft version that cannot interact with Ds, and PCP defects in ds mutants can be rescued by uniform overexpression of Ds. Moreover, blocking overgrowth through removal of dachs also suppresses PCP phenotypes in both mutants. The remaining disturbances in PCP in each of these backgrounds are restricted to very proximal regions, both in adult wings and the wing disc. Thus, the graded distribution of Ds does not provide a direct cue to orient PCP over long distances; rather, it appears to be important only locally near the hinge. Furthermore, this study shows that the two other key signaling pathways that contribute to the global PCP pattern in the disc do not act directly through long-range gradients. How do these signals specify the PCP pattern, if not through gradients (Sagner, 2012)?

Simulations in the vertex model have suggested that long-range polarity can be established in the absence of global biasing cues if PCP is allowed to develop during growth. PCP easily aligns in a small system, and globally aligned polarity can then be maintained as the system grows. Such a model obviates the necessity of long-range biasing cues like gradients, at least to maintain long-range alignment of PCP domains. The finding that a global PCP pattern develops early during growth of the wing makes this idea plausible. It may be that a combination of local signals at the different organizer regions specifies the vector orientation of PCP when the disc is still small, and that the pattern is maintained during growth. This may explain why loss-of-function studies have failed to identify the signaling pathways at the AP and DV boundaries as important organizers of the PCP pattern (Sagner, 2012).

In addition to local signals, the orientation of growth may provide additional cues that help shape the PCP pattern. Simulating the interplay between PCP and growth in the vertex model showed that oriented cell divisions and cell rearrangements orient PCP either parallel or perpendicular to the axis of tissue elongation, depending on parameters. Interestingly, each of the signaling pathways that influence PCP in the disc also influences the disc growth pattern. Wg/Notch signaling at the DV boundary drives growth parallel to the DV boundary, consistent with the pattern of clone elongation at the DV boundary. Growth near the AP boundary, where Hh signaling is most active, is oriented parallel to the AP boundary. This behavior probably reflects oriented cell rearrangements rather than oriented cell divisions. Finally, Ft and Ds orient growth away from the hinge. Suppressing overgrowth in ft or ds mutant wings by altering downstream components of the Hippo pathway rescues normal PCP except in the most proximal regions of the wing. Thus, altered growth orientation may contribute to the PCP defects seen in ft and ds mutants (Sagner, 2012).

Growth orientation reflects the orientation of both cell divisions and neighbor exchanges, and these can each exert different effects on the axis of PCP. Understanding the influence of local growth patterns on PCP will require a quantitative description of the patterns of cell divisions and rearrangements in the disc. More refined simulations incorporating local differences in the orientation of cell divisions and rearrangements will allow exploration of how planar polarity patterns can be guided by different growth patterns (Sagner, 2012).

Wingless and the synapse

Heterotrimeric Go protein links Wnt-Frizzled signaling with ankyrins to regulate the neuronal microtubule cytoskeleton

Drosophila neuromuscular junctions (NMJs) represent a powerful model system with which to study glutamatergic synapse formation and remodeling. Several proteins have been implicated in these processes, including components of canonical Wingless (Drosophila Wnt1) signaling and the giant isoforms of the membrane-cytoskeleton linker Ankyrin 2, but possible interconnections and cooperation between these proteins were unknown. This study demonstrates that the heterotrimeric G protein Go functions as a transducer of Wingless-Frizzled 2 signaling in the synapse. Ankyrin 2 was identified as a target of Go signaling required for NMJ formation. Moreover, the Go-ankyrin interaction is conserved in the mammalian neurite outgrowth pathway. Without ankyrins, a major switch in the Go-induced neuronal cytoskeleton program is observed, from microtubule-dependent neurite outgrowth to actin-dependent lamellopodial induction. These findings describe a novel mechanism regulating the microtubule cytoskeleton in the nervous system. This work in Drosophila and mammalian cells suggests that this mechanism might be generally applicable in nervous system development and function (Luchtenborg, 2014).

Ankyrins (Ank) are highly abundant modular proteins that mediate protein-protein interactions, mainly serving as adaptors for linking the cytoskeleton to the plasma membrane. Mammalian genomes encode three Ank genes [AnkR (Ank1), AnkB (Ank2) and AnkG (Ank3)], whereas Drosophila has two [Ank1 (also known as Ank - FlyBase) and Ank2]. Ank2 is expressed exclusively in neurons and exists in several splicing variants. The larger isoforms (Ank2M, Ank2L and Ank2XL) are localized to axons and play important roles in NMJ formation and function. The C-terminal part of Ank2L can bind to microtubules. Despite the well-established role of Ank2 in NMJ formation, its function has been considered somewhat passive and its mode of regulation has not been clarified. This study shows that Gαo binds to Ank2 and that these proteins and the Wg pathway components Wg, Fz2, and Sgg jointly coordinate the formation of the NMJ. The functional Gαo-Ank interaction is conserved from insects to mammals (Luchtenborg, 2014).

Synaptic plasticity underlies learning and memory. Both in invertebrates and vertebrates, activation of Wnt signaling is involved in several aspects of synapse formation and remodeling, and defects in this pathway may be causative of synaptic loss and neurodegeneration. Thus, understanding the molecular mechanisms of synaptic Wnt signaling is of fundamental as well as medical importance. The Drosophila NMJ is a powerful model system with which to study glutamatergic synapses, and the Wnt pathway has been widely identified as one of the key regulators of NMJ formation.

This study provides important mechanistic insights into Wnt signal transduction in the NMJ, identifying the heterotrimeric Go protein as a crucial downstream transducer of the Wg-Fz2 pathway in the presynapse. It was further demonstrated that Ank2, a known player in the NMJ, is a target of Gαo in this signaling (Luchtenborg, 2014).

This study found that the α subunit of Go is strongly expressed in the presynaptic cell, and that under- or overactivation of this G protein leads to neurotransmission and behavioral defects. At the level of NMJ morphology, presynaptic downregulation or Ptx-mediated inactivation of Gαo was found to recapitulate the phenotypes obtained by similar silencing of wg and fz2. These data confirm that presynaptic Wg signaling, in addition to the Wg pathway active in the muscle, is crucial for proper NMJ formation, and that Go is required for this process. Furthermore, neuronal Gαo overexpression can rescue the wg and fz2 loss-of-function phenotypes, demonstrating that, as in other contexts of Wnt/Fz signaling, Go acts as a transducer of Wg/Fz2 in NMJ formation. In contrast to its evident function and clear localization in the presynapse, Gαo localization on the muscle side of the synapse is much less pronounced or absent. Unlike Gαo, the main Drosophila Gβ subunit is strongly expressed in both the pre- and postsynapse. Thus, a heterotrimeric G protein other than Go might be involved in the postsynaptic Fz2 transduction, as has been implicated in Fz signaling in some other contexts (Luchtenborg, 2014).

A recent study proposed a role for Gαo downstream of the octopamine receptor Octβ1R. This signaling was proposed to regulate the acute behavioral response to starvation both on type II NMJs (octapaminergic) and on the type I NMJs (glutamatergic) analyzed in this study. In contrast to the current observations, downregulation of Gαo in these NMJs was proposed to increase, rather than decrease, type I bouton numbers. It is suspected that the main reason for the discrepancy lies in the Gal4 lines used. The BG439-Gal4 and C380-Gal4 lines of Koon are poorly characterized and, unlike the well-analyzed pan-neuronal elav-Gal4 and motoneuron-specific OK371-Gal4 and D42-Gal4 driver lines used in the current study, might mediate a more acute expression. In this case, this study reflects the positive role of Gαo in the developmental formation of glutamatergic boutons, as opposed to a role in acute fine-tuning in response to environmental factors as studied by Koon (Luchtenborg, 2014).

Postsynaptic expression of fz2 was found to fully rescue fz2 null NMJs. This study found that presynaptic knockdown of Fz2 (and other components of Wg-Fz2-Gαo signaling) recapitulates fz2 null phenotypes, whereas presynaptic overactivation of this pathway increases bouton numbers; furthermore, presynaptic overexpression of fz2 or Gαo rescues the fz2 nulls, just as postsynaptic overexpression of fz2 does. The current data thus support a crucial role for presynaptic Wg-Fz2-Gαo signaling in NMJ formation. Interestingly, both pre- and postsynaptic re-introduction of Arrow, an Fz2 co-receptor that is normally present both pre- and postsynaptically, as is Fz2 itself, can rescue arrow mutant NMJs. Thus, it appears that the pre- and postsynaptic branches of Fz2 signaling are both involved in NMJ development. A certain degree of redundancy between these branches must exist. Indeed, wild-type levels of Fz2 in the muscle are not sufficient to rescue the bouton defects induced by presynaptic expression of RNAi-fz2, yet overexpression of fz2 in the muscle can restore the bouton integrity of fz2 nulls. One might hypothesize that postsynaptic Fz2 overexpression activates a compensatory pathway - such as that mediated by reduction in laminin A signaling - that leads to restoration in bouton numbers in fz2 mutants. The current data showing that the targeted downregulation of Fz2 in the presynapse is sufficient to recapitulate the fz2 null phenotype underpin the crucial function of presynaptic Fz2 signaling in NMJ formation (Luchtenborg, 2014).

This study found that downregulation of Ank2 produces NMJ defects similar to those of wg, fz2 or Gαo silencing. However, Ank2 mutant phenotypes appear more pronounced, indicating that Wg-Fz2-Gαo signaling might control a subset of Ank2-mediated activities in the NMJ. Ank2 was proposed to play a structural role in NMJ formation, binding to microtubules through its C-terminal region. However, since the C-terminal region was insufficient to rescue Ank2L mutant phenotypes, additional domains are likely to mediate Ank2 function through binding to other proteins. This study demonstrates in the yeast two-hybrid system and in pull-down experiments that the ankyrin repeat region of Ank2 physically binds Gαo, suggesting that the function of Ank2 in NMJ formation might be regulated by Wg-Fz2-Gαo signaling. Indeed, epistasis experiments place Ank2 downstream of Gαo in NMJ formation (Luchtenborg, 2014).

Upon dissociation of the heterotrimeric Go protein by activated GPCRs such as Fz2, the liberated Gαo subunit can signal to its downstream targets both in the GTP- and GDP-bound state (the latter after hydrolysis of GTP and before re-association with Gβγ). The free signaling Gαo-GDP form is predicted to be relatively long lived, and a number of Gαo target proteins have been identified that interact equally well with both of the nucleotide forms of this G protein. In the context of NMJ formation, this study found that Gαo-GTP and -GDP are efficient in the activation of downstream signaling, and identifies Ank2 as a binding partner of Gαo that interacts with both nucleotide forms. The importance of signaling by Gα-GDP released from a heterotrimeric complex by the action of GPCRs has also been demonstrated in recent studies of mammalian chemotaxis, planar cell polarity and cancer (Luchtenborg, 2014).

Gαo[G203T], which largely resides in the GDP-binding state owing to its reduced affinity for GTP, might be expected to act as a dominant-negative. However, in canonical Wnt signaling, regulation of asymmetric cell division as well as in planar cell polarity (PCP) signaling in the wing, Gαo[G203T] displays no dominant-negative activity but is simply silent, whereas in eye PCP signaling this form acts positively but is weaker than other Gαo forms. Biochemical characterization of the mammalian Gαi2[G203T] mutant revealed that it can still bind Gβγ and GTP, but upon nucleotide exchange Gαi2[G203T] fails to adopt the activated confirmation and can further lose GTP. The current biochemical characterization confirms that Gαo[G203T] still binds GTP. Interestingly, Gαi2[G203T] inhibited only a fraction of Gαi2-mediated signaling, suggesting that the dominant-negative effects of the mutant are effector specific. Thus, it is inferred that a portion of Gαo[G203T] can form a competent Fz2-transducing complex, and a portion of overexpressed Gαo[G203T] resides in a free GDP-loaded form that is also competent to activate downstream targets - Ank2 in the context of NMJ formation (Luchtenborg, 2014).

These experiments place Ank2 downstream of Gαo and also of Sgg (GSK3β). It remains to be investigated whether Ank2 can directly interact with and/or be phosphorylated by Sgg. Meanwhile, it is proposed that the microtubule-binding protein Futsch might be a linker between Sgg and Ank2. Futsch is involved in NMJ formation and is placed downstream of Wg-Sgg signaling, being the target of phosphorylation and negative regulation by Sgg as the alternative target to β-catenin, which is dispensable in Wg NMJ signaling. Abnormal Futsch localization has been observed in Ank2 mutants. In Drosophila wing and mammalian cells in culture, Gαo acts upstream of Sgg/GSK3β. Cumulatively, these data might suggest that the Wg-Fz2-Gαo cascade sends a signal to Futsch through Sgg, parallel to that mediated by Ank2 (Luchtenborg, 2014).

The importance of the Gαo-Ank2 interaction for Drosophila NMJ development is corroborated by findings in mammalian neuronal cells, where it was demonstrated that the ability of Gαo to induce neurite outgrowth is critically dependent on AnkB and AnkG. Knockdown of either or both ankyrin reduces neurite production. Remarkably, upon AnkB/G downregulation, Gαo switches its activity from the induction of microtubule-dependent processes (neurites) to actin-dependent protrusions (lamellopodia). Furthermore, Gαo recruits AnkB to the growing neurite tips. These data demonstrate that the Gαo-ankyrin mechanistic interactions are conserved from insects to mammals and are important for control over the neuronal tubulin cytoskeleton in the context of neurite growth and synapse formation. The novel signaling mechanism that were uncovered might thus be of general applicability in animal nervous system development and function (Luchtenborg, 2014).

Table of contents

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

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