Effects of Mutation or Deletion (part 2/2)

Dishevelled and tissue polarity

Polarized cells in all body segments require dishevelled function to establish planar cell polarity. The requirement for dishevelled in establishing polarity is cell autonomous (Theisen, 1994).

Three genes, frizzled, dishevelled, and prickle, share a similar tissue polarity phenotype, suggesting that they function together in a single process. dsh encodes a dosage sensitive component required for fz function and it likely acts downstream of fz in the generation of tissue polarity. These findings suggest that DSH may have a general role in signal transduction, perhaps as a component of a receptor complex (Krasnow, 1995).

The tissue polarity genes of Drosophila are required for correct establishment of planar polarity in epidermal structures, which in the eye is shown in the mirror-image symmetric arrangement of ommatidia relative to the dorsoventral midline. Mutations in the genes frizzled, dishevelled and prickle-spiny-legs (pk-sple) result in the loss of this mirror-image symmetry. fz encodes a serpentine receptor-like transmembrane protein required for reception and transmission of a polarity signal. Little else is known of the signaling pathway(s) involved other than that Dsh acts downstream of Fz. Mutations have been identified in the Drosophila homolog of Rho1 p21 GTPase; by analysis of their phenotypes it has been shown that Rho1 is required for the generation of tissue polarity. Genetic interactions indicate a role for Rho1 in signaling mediated by Fz and Dsh, and suggest that JNK/SAPK-like kinases are involved. These data are consistent with a Fz/Rho1 signaling cascade analogous to the yeast pheromone signaling pathway and that proposed for activation of the serum response factor (SRF) in vertebrate cells (Strutt, 1997).

During metazoan development, cell-fate diversity is brought about, in part, by asymmetric cell divisions. In Drosophila, bristle mechanosensory organs are composed of four different cells that originate from a single precursor cell (pI) after two rounds of asymmetric division. At each division, distinct fates are conferred on sister cells by the asymmetric segregation of Numb, a negative regulator of Notch signaling. The orientation of the mitotic spindles and the localization of the Numb crescent follow a stereotyped pattern. Mitosis of pI is oriented parallel to the anteroposterior axis of the fly. In all cases, Numb is distributed in an anterior crescent, and is segregated to the anterior daughter cell. The posterior daughter cell, pIIa, divides to generate the shaft and socket cells, and the anterior daughter cell, pIIb, divides to give rise to the neuron and the sheath cells. In all cases Numb accumulates in the anterior pole of pIIa, next to pIIb, and segregates to the anterior daughter, which differentiates into a shaft cell. pIIb divides soon after pIIa. Mitotic spindles of pIIb are oriented roughly orthogonal to the previous pIIa division axis. Numb is localized at the lateral pole, that is, away from the midline of the pIIb cell. As the lateral daughter cell inherits Numb, it is predicted that the lateral cell adopts a neuronal fate. The pattern of oriented mitosis is probable essential for sensory functions. Changing the identity of pIIb into a second pIIa by ectopic Notch signaling results in a second pIIa, which orients itself in a similar position to that of the original pIIa (Gho, 1998).

Signalling mediated by the Frizzled receptor polarizes pI along the A/P axis, thereby specifying the orientation of the mitotic spindle and positioning the Numb crescent. Mitoses in fz and dishevelled result in randomly oriented pI divisions in the epithelial plane. The Numb crescent also localizes in a random manner, however, its position is tightly correlated with the position of one pole of the misoriented spindle. Only pI cells respond to Fz/Dsh signaling. It is concluded that the polarity of the three mitotic cells in the bristle lineage are regulated by distinct mechanisms. Inscuteable is unlikely to be the pI organizer because clonal analysis shows that insc regulates neither bristle differentiation nor polarity in the notum. Thus the organizer acting downstream of Fz signaling (upstream of Numb) in planar divisions remains to be identified (Gho, 1998).

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. 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).

Epistasis analysis was carried out to position Adenomatous polypopsis coli tumor suppressor homolog 2 (Apc2) with respect to other components of the signal transduction pathway. wg; Apc2DeltaS double mutant embryos (with Apc2DeltaS mutant mothers) show a partial rescue of the wg phenotype, with restoration of the normal diversity of cuticular pattern elements and small expanses of naked cuticle, suggesting that Apc2 is downstream of wg. There are two possible explanations for the fact that the double mutant does not show the same phenotype as the Apc2 single mutant: either Apc2DeltaS is not null, or the negative regulatory machinery remains partially active in the absence of Apc2. If Apc2DeltaS is not null, it was reasoned that repeating the epistasis test with Apc2DeltaS in trans to a deficiency removing Apc2 (Df(3R)crb87-4) might further reduce Apc2 function, producing a double mutant phenotype more similar to that of Apc2DeltaS alone. However, when this was done, there was no change in the double mutant phenotype, suggesting that Apc2DeltaS may be genetically null for this function. Other components of the Wg signal transduction pathway act downstream of Apc2. Embryos maternally and zygotically mutant for both dishevelled (dsh) and Apc2 show a phenotype indistinguishable from the dsh single mutant, as do embryos maternally mutant for both dsh and Apc2 that are zygotically dsh/Y; Apc2DeltaS/Df(3R)crb87-4. Likewise, arm; Apc2 and Apc2; dTCF double mutants (derived from Apc2 homozygous mothers) are indistinguishable from arm or dTCF single mutants. Thus, dsh, arm, and dTCF all act genetically downstream of Apc2; this was expected for arm and dTCF, but was surprising for dsh (McCartney, 1999)

In an attempt to pursue functional relationships between starry night/flamingo and previously discovered tissue polarity genes, a study was carried out to see whether Stan distribution is altered in various polarity mutants, particularly in fz complete loss-of-function mutants. In the total absence of Fz protein (fzD21/fzK21) Stan is not redistributed, as it normally is, toward the proximal/distal (P/D) cellular boundaries at 24 or 30 hr after puparium formation (APF). At the onset of prehair formation (30 hr APF), bright staining at cell boundaries is greatly reduced in length, and the fragmented signals are not necessarily restricted to the P/D boundaries, indicating that generation of the normal Stan pattern is strongly dependent on Fz. Residual boundary signals become even less prominent at later stages, leaving only fine dots both in the cytoplasm and along cell borders. Along the apicobasal cell axis, these intracellular particles are present from near the apical surface to the basolateral level. dsh1 (an allele of dishevelled) is a genetic null allele for planar polarity. Wings with this mutation also show a decrease in intensity of Stan staining at cell boundaries, and the distribution appears to be much less polarized than that in wild type. Mutations of other genes involved in tissue polarity do not necessarily disrupt the Stan distribution. For example, in mutant cells of the multiple wing hair (mwh) gene, which is currently considered to be further downstream in the tissue polarity pathway, Stan molecules are present predominantly at P/D boundaries. Therefore, Fz and one downstream component, Dsh, are thought to be necessary to accomplish the normal distribution of Stan (Usui, 1999).

The Drosophila eye, a paradigm for epithelial organization, is highly polarized with mirror-image symmetry about the equator. The R3 and R4 photoreceptors in each ommatidium are vital in this polarity; they adopt asymmetrical positions in adult ommatidia and are the site of action for several essential genes. Two such genes are frizzled (fz) and dishevelled (dsh), the products of which are components of a signaling pathway required in R3, and which are thought to be activated by a diffusible signal. The transmembrane receptor Notch is required downstream of dsh in R3/R4 for them to adopt distinct fates. By using an enhancer for the Notch target gene Enhancer of split mdelta, it is shown that Notch becomes activated specifically in R4. Analyzing the regulation of E(spl)mdelta, it has been found that this target of Notch is expressed specifically in R4. Transiently reducing Notch activity for 6 hours in late-third-instar larvae, using temperature sensitive Notch, leads to a loss of E(spl)mdelta expression, whereas transient activation of a constitutively active Notch derivitive has the converse effect. Genetic experiments show the importance of Dsh in the establishment of eye polarity. The mutant protein coded for in dishevelled mutants has impaired signaling, but the phenotype can be partially rescued by overexpressing downstream components. When E(spl) proteins are expressed ectopically in dsh mutant discs, the eyes are less roughened and more ommatidia have correct rotation and chirality, compared to when E(spl) proteins are not ectopically expressed. It is proposed that Fz/Dsh promotes expression of the Notch ligand Delta and inhibits Notch receptor activity in R3, creating a difference in Notch signaling capacity between R3 and R4. Subsequent feedback in the Notch pathway ensures that this difference becomes amplified. This interplay between Fz/Dsh and Notch indicates that polarity is established through local comparisons between two cells and explains how a signal from one position (for example, the equator in the eye) could be interpreted by all ommatidia in the field. Additional targets of Notch needed to specify R4 identity may include strabismus. Ommatidial polarity also requires spiney-legs (sple). Sple is normally inhibitory to Notch in R3, because high levels of E(spl)mdelta expression are found in sple mutants. The inhibitory effect of Sple provides a second mechanism for polarizing Notch signaling in R3/R4 that could be coordingated by Fz/Dsh (Cooper, 1999).

Planar polarity is seen in epidermally derived structures throughout the animal kingdom. In the Drosophila eye, planar polarity is reflected in the mirror-symmetric arrangement of ommatidia (eye units) across the dorsoventral midline or equator; ommatidia on the dorsal and ventral sides of the equator exhibit opposite chirality. Photoreceptors R3 and R4 are essential in the establishment of the polarity of ommatidia. The R3 cell is thought to receive the polarizing signal, eminating from the equator, through the receptor Frizzled (Fz), before or at higher levels than the R4 cell, generating a difference between neighbouring R3 and R4 cells. Both loss-of-function and overexpression of Fz in the R3/R4 pair result in polarity defects and loss of mirror-image symmetry. Notch and Delta (Dl) are identified as dominant enhancers of the phenotypes produced by overexpression of fz and dishevelled (dsh); dsh encodes a signaling component downstream of Fz, and it is shown that Dl-mediated activation of Notch is required for establishment of ommatidial polarity. Whereas fz signaling is required to specify R3, Notch signaling induces the R4 fate. These data indicate that Dl is a transcriptional target of Fz/Dsh signaling in R3, and Dl activates Notch in the neighboring R4 precursor. This two-tiered mechanism explains how small differences in the level and/or timing of Fz activation reliably generate a binary cell-fate decision, leading to specification of R3 and R4 and ommatidial chirality. How Notch signaling induces the R4 fate remains unclear, as it usually represses photoreceptor development at this stage. However, the precursor cells are already committed to form the R3/R4 pair by transcription factors (such as Seven-up) that are required for both R3/R4-cell fate and polarity generation (Fanto, 1999).

The small GTPases Rac and Rho act as cellular switches in many important biological processes. In the fruit fly Drosophila, RhoA participates in the establishment of planar polarity, a process mediated by the receptor Frizzled (Fz). Thus far, analysis of Rac in this process has not been possible because of the absence of mutant Rac alleles. The roles of Rac and Rho in establishing the polarity of ommatidia in the Drosophila eye were investigated. By expressing a dominant negative or a constitutively activated form of Rac1, Rac signaling was interfered with specifically and ommatidial polarity was disrupted. The resulting defects are similar to the loss/gain-of-function phenotypes typical of tissue-polarity genes. Through genetic interaction and rescue experiments involving a polarity-specific, loss-of-function dishevelled (dsh) allele, Rac1 was found to act downstream of Dsh in the Fz signaling pathway, but upstream of, or in parallel to, RhoA. Rac signals to the nucleus through the Jun N-terminal kinase (JNK) cascade in this process. By generating point mutations in the effector loop of RhoA, it was found that RhoA also signals to the nucleus during the establishment of ommatidial polarity. Nevertheless, Rac and RhoA activate transcription of distinct target genes. Thus Rac is specifically required downstream of Dsh in the Fz pathway. It functions upstream or in parallel to RhoA and both signal to the nucleus, through distinct effectors, to establish planar polarity in the Drosophila eye (Fanto, 2000).

Frizzled and the wingless pathway are involved in tissue polarity in Drosophila. A method was devised to induce a gradient of fz expression with the highest levels near the distal wing tip. The result is a large area of proximally pointing hairs in this region. This reversal of polarity is seen when fz expression is induced just before the start of hair morphogenesis, at the time polarity is established, suggesting that the gradient of Fz protein acts fairly directly to reverse hair polarity. A similar induction of the dishevelled (dsh) gene, which acts cell autonomously and functions downstream of fz in the generation of tissue polarity, results in a distinct tissue polarity phenotype, but indicates no reversal of polarity; this argues that fz signaling is required for polarity reversal. The finding that functional dsh is required for the reversal of polarity argues that the reversal requires normal fz signal transduction. The data suggest that cells sense the level of Fz protein on neighboring cells and use this information in order to polarize themselves. A polarizing signal is transmitted from cells with higher Fz levels to cells with lower levels. These observations enable the proposal of a general mechanism to explain how Wnts polarize target cells (Adler, 1997).

The adult cuticular wing of Drosophila is covered by an array of distally pointing hairs, revealing the planar polarity of the wing. Mutations in dachsous disrupt this regular pattern by affecting frizzled signaling. dachsous encodes a large membrane protein that contains many cadherin domains and dachsous mutations cause deformed body parts. Mutations in dachsous also result in a tissue polarity phenotype that is similar to frizzled, dishevelled and prickle at the cellular level, because many cells form a single hair of abnormal polarity. Although the cellular phenotype is similar to frizzled, dishevelled and prickle, dachsous mutant wings display a unique and distinctive abnormal hair polarity pattern including regions of reversed polarity. The development of this pattern requires the function of frizzled pathway genes, suggesting that in a dachsous mutant the frizzled pathway is functioning - but in an abnormal way. Genetic experiments indicated that dachsous is not required for the intracellular transduction of the frizzled signal. However, dachsous clones disrupt the polarity of neighboring wild-type cells, suggesting the possibility that dachsous affects the intercellular signaling function of frizzled. Consistent with this hypothesis, frizzled clones in a dachsous mutant background display enhanced domineering non-autonomy, and the anatomical direction of this domineering non-autonomy is altered in regions of dachsous wings with abnormal hair polarity. The direction of this domineering nonautonomy is coincident with the direction of the abnormal hair polarity. It is concluded that dachsous causes a tissue polarity phenotype because it alters the direction of frizzled signaling. Perhaps ds mutations alter the efficiency, stability or propagation of a polarity signal in a way that leads to the system becoming destabilized. The regions of reversed polarity might be caused by minor perturbations in fz signaling being amplified and propagated in a ds mutant wing due to decreased stability of the system. It is possible that fz signaling takes place at the adherens junction and that ds mutations alter the structure or composition of the junction in a way that alters fz signaling (Adler, 1998).

Misshapen acts in the Frizzled (Fz) mediated epithelial planar polarity (EPP) signaling pathway in eyes and wings. Both msn loss- and gain-of-function result in defective ommatidial polarity and wing hair formation. Genetic and biochemical analyses indicate that msn acts downstream of fz and dishevelled (dsh) in the planar polarity pathway, and thus implicates an STE20-like kinase in Fz/Dsh-mediated signaling. This demonstrates that seven-pass transmembrane receptors can signal via members of the STE20 kinase family in higher eukaryotes. Msn acts in EPP signaling through the JNK (Jun-N-terminal kinase) module as it does in dorsal closure. Although at the level of Fz/Dsh there is no apparent redundancy in this pathway, the downstream effector JNK/MAPK (mitogen-activated protein kinase) module is redundant in planar polarity generation. To address the nature of this redundancy, evidence is provided for an involvement of the related MAP kinases of the p38 subfamily in planar polarity signaling downstream of Msn (Paricio, 1999).

In the Drosophila eye, EPP is reflected in the mirror-symmetric arrangement of ommatidial units relative to the dorso-ventral midline (the equator). This pattern is generated posterior to the morphogenetic furrow when ommatidial preclusters rotate 90° toward the equator, adopting opposite chirality depending on their dorsal or ventral positions. Polarity defects are manifested in the loss of mirror-image symmetry, with the ommatidia misrotating and adopting random chirality or remaining symmetrical. The gain-of-function dsh phenotype (sev-Dsh) has been successfully used in previous reports to identify new components of the Fz/Dsh planar polarity pathway. This same assay, dominant genetic modification of the sev-Dsh phenotype, was used to screen through a large number of known genes. Among the few mutants that show a specific interaction are two msn alleles. msn102 and msn172 are X-ray-induced inversions with breakpoints in the msn gene. Both loss-of-function alleles of msn act as dominant suppressors of sev-Dsh, comparable to other planar polarity-specific Dsh effectors (Paricio, 1999).

In addition, msn has been isolated in a gain-of-function screen for genes involved in planar polarity generation. Overexpression of genes required in planar polarity signaling at the relevant time often results in defects that are similar to the loss-of-function mutant phenotypes, e.g. with Fz and Dsh. In such a screen, ap-GAL4 flies (ap-GAL4 induces overexpression of the corresponding gene in the notum and the dorsal part of the wing), were crossed to the collection of 2200 E/P lines and the progeny were scored for disarranged microchaetae on the notum. One of the lines isolated in this screen, ep(3)0549, shows an abnormal orientation of the microchaetae similar to phenotypes obtained with ap driven Fz overexpression. Similarly, ap-GAL4, ep(3)0549 flies show typical polarity phenotypes on the dorsal surface of the wing where these are manifest in the presence of multiple wing hairs. In situ hybridization experiments to polytene chromosomes and complementation analyses reveal that the EP-element insertion in line ep(3)0549 is in the msn locus and represents a msn allele. Subsequent sequence analyses confirm that the EP insertion is located 24 bp upstream of the 5'-end of a msn cDNA. Taken together, these results suggest that msn is involved in EPP signaling and possibly acts downstream of Dsh (Paricio, 1999).

To gain further confirmation of the role of Msn in Fz/Dsh-mediated polarity signaling, an in vitro assay was used to determine whether Msn acts downstream of Dsh in JNK pathway activation. Previous experiments have shown that expression of Dsh in NIH 3T3 cells activates JNK and Jun phosphorylation, indicating that Dsh is a potent activator of a Jun-kinase pathway. Using the same assay, it was asked whether co-expression of a dominant-negative (kinase-inactive) Msn protein (DN-Msn) has an effect on Dsh-induced Jun phosphorylation. Significantly, co-expression of DN-Msn in this context causes a dramatic concentration-dependent inhibition of Jun phosphorylation. Taken together with the genetic interactions, these experiments confirm that Msn is acting downstream of Fz/Dsh in planar polarity signaling (Paricio, 1999).

msn mutations affect the morphology of the rhabdomeres in photoreceptors, causing malformed, 'misshapen' rhabdomeres, and also, at lower frequency, the number of photoreceptors. In addition, msn is required for the process of dorsal closure, and embryos mutant for msn display a typical dorsal open phenotype. To analyze its requirements in polarity generation, msn mutant clones in the eye and the wing were examined in detail. A phenotypic analysis of eye clones reveals that msn is required for the generation of planar polarity. msn mutant ommatidia containing the normal complement of photoreceptors are often misrotated and display the wrong chiral form or are symmetrical (non-chiral). To confirm that the polarity defects of msn mutant ommatidia are primary defects, and thus implicate msn in polarity generation, ommatidial polarity was examined in msn mutant clones at the earliest possible stage in third instar larval imaginal discs (when tissue polarity genes are required). Spalt is expressed in the R3/R4 precursor pair for about two columns at this stage. In wild type this reflects the regular arrangement and direction of rotation of the preclusters. In msn mutant tissue, ommatidial rotation, and thus polarity, is randomized (e.g. ommatidia rotate in the opposite direction as their wild-type neighbors) showing that these defects result from an early failure in polarity establishment. Thus in the eye, the msn phenotype (defects in polarity, malformed, misshapen and missing photoreceptors) is very reminiscent of other genes involved in both polarity and terminal photoreceptor differentiation (Paricio, 1999).

The fz gene has been implicated in the specification of the R3 cell within the R3/R4 pair in the process of chirality generation. The mosaic analysis of both loss-of-function and gain-of-function fz alleles has shown that Fz signaling is required in R3 for correct ommatidial chirality generation and also induces R3 fate. The genetic interactions and cell culture experiments have shown that msn acts downstream of Fz/Dsh, and thus it was asked whether msn is also involved in the selection of R3 in analogy to the fz requirement. The genotypic composition of mosaic ommatidial clusters were examined within the R3/R4 pair. This analysis revealed that, as is the case for fz, the msn+ cell has a strong preference for adopting the R3 photoreceptor fate. This can often lead to chirality inversions, where the msn+ R4 precursor adopts the R3 position and displaces the original msn- R3 precursor. In summary, the genetic requirements of msn in single photoreceptors, in particular the R3/R4 pair, are very similar to those of fz (Paricio, 1999).

Deletion of the entire prickle gene gives a phenotype over the whole body surface that is similar to dsh and fz. To test whether these mutants affect the same signaling pathway, double mutant combinations were made. Double mutants of pkpk and dsh in the triple row give a Dsh phenotype, suggesting that dsh is epistatic to pkpk. In other words, dsh function is blocked; pk function is irrelevant. These data suggest that many of the functions of pk require dsh. The situation within the wing blade is less clear, as the double mutants give an intermediate phenotype. The dsh; pksple double mutant retains a dsh phenotype in the triple row, but the wing hair pattern is altered, despite the fact that pksple alleles have no wing phenotype. In the leg, the dsh tarsal phenotype is not modified by pkpk. There is a synergistic interaction between dsh and pksple, with the double mutant giving a more extreme mirror-image transformation of the T1 segment than either of the single mutants. This extreme transformation is also seen with UAS:pk+ overexpression and in fz (Gubb, 1999).

The tarsal phenotype of dsh is very similar to pkpksple13 causing duplications of the T3 and T4, segments. A more extreme phenotype including complete duplications of T2 to T4 together with a well-developed ectopic T1 joint are seen in dsh; pksple, overexpression UAS:pk+ and fz mutant strains, implying that a similar polarity signaling pathway is affected. It has been suggested that pk and fz are upstream in a signaling pathway leading to dsh (Wong, 1993 and Shulman, 1998). There are several problems with a simple linear pathway, however, and the relationship between pk and fz is unclear. (1) Neither fz nor pk is clearly epistatic to the other; rather, the double mutant (pkpk; fz) phenotype is intermediate between pkpk and fz (Gubb, 1982 and Wong, 1993). (2) Whereas fz clones cause a long-range nonautonomous disruption in surrounding tissue, pkpk clones resemble dsh clones in being almost completely cell autonomous. (Both pksple1 and pkpk-sple13 clones are cell autonomous in the eye. In contrast, the pkpk triple row bristle phenotype (that results from Sple expression in the absence of Pk) is suppressed in dsh; pkpk wings and the dsh polarity pattern is modified in dsh; pksple wings (despite pksple on its own not having a wing phenotype). (3) Complete lack of pk transcripts produces a phenotype very similar to dsh. Taken together, these results indicate that Pk is not downstream of Fz but may represent an alternative input into Dsh-mediated planar polarity signaling. If Dsh is acting as a scaffolding or adapter protein then it would be required in stoichiometric ratios to its target proteins, and overexpression would prevent assembly of functional complexes. It may be that the Pk protein isoforms are components of this protein complex that are expressed in cells remodeling their cytoskeletal architecture (Gubb, 1999).

expanded plays a role in patterning of the eye, mainly at the level of planar polarity. Mutant exe1 clones exhibit penetrant phenotypes that are characterized by ommatidial chirality inversions, misrotations, and minor defects in photoreceptor differentiation. In order to determine whether these defects arise during early stages of development, an antibody against the Spalt protein, a marker for the R3 and R4 photoreceptor precursors (on which ommatidial rotation and chirality depend) was used to analyze rotation defects within mutant tissue in pupal imaginal discs. The random orientation of many of the R3/R4 photoreceptor precursor pairs within the clone at this early stage indicates that defects in planar polarity are likely to be a primary consequence of loss of ex function. ex does not affect the initiation of differentiation or the progression of the morphogenetic furrow (differentiation is evident in both imaginal disc and adult tissue), but ex does play a role in orchestrating the fine details of the ensuing cell fate specification and planar polarization events (Blaumueller, 2000).

Given the planar polarity phenotype of ex loss-of-function mutants, a test was made of the ability of ex alleles to genetically interact with gain-of-function genotypes generated by overexpression of components of the frizzled (fz) planar polarity pathway. The gain-of-function frizzled and disheveled phenotypes (sev-fz, sev-dsh), and also sev-rhoAV14 and sev-racV12, have been successfully used in previous studies both to identify new components of the Fz/Dsh planar polarity pathway, and to genetically position known components with respect to others. This assay, dominant genetic modification of sev-fz, sev-dsh, sev-rhoAV14 and sev-racV12, was used to analyze in more detail the role of Ex in this process. These experiments did not reveal significant genetic interactions between ex and most of these genotypes. However, sev-dsh is dominantly enhanced by exe1, with an increase of ommatidia that are unscorable with respect to polarity because they lack one or more photoreceptors. This phenotypic modification could be achieved by several mechanisms, and is suggestive of complex cross-talk, either between multiple signaling pathways (dsh itself plays a role in multiple signaling pathways, including those represented by wingless, frizzled, and possibly Notch), or between signaling pathways and the mechanical processes required to carry out the instructions provided by the signaling pathways (Blaumueller, 2000).

Rho-associated kinase works downstream of Fz/Dsh to mediate a branch of the planar polarity pathway involved in ommatidial rotation in the eye

Drosophila Rho-associated kinase (Rok) works downstream of Fz/Dsh to mediate a branch of the planar polarity pathway involved in ommatidial rotation in the eye and in restricting actin bundle formation to a single site in developing wing cells. The primary output of Rok signaling is regulating the phosphorylation of nonmuscle myosin regulatory light chain (Mizuno, 1999 and Winter, 2001), and hence the activity of myosin II. Drosophila myosin VIIA, the homolog of the human Usher Syndrome 1B gene, also functions in conjunction with this newly defined portion of the Fz/Dsh signaling pathway to regulate the actin cytoskeleton (Winter, 2001).

The similarity of the Drok2 clonal phenotype in the wing to aspects of the phenotypes of fz, dsh, and RhoA led to the hypothesis that Rok may act downstream of Fz/Dsh. To assess the genetic interactions among these genes, the multiple hair phenotype was quantitated in a defined region: the ventral surface of the proximal-anterior region of the wing. Use was made of the dsh1 allele, which is defective for PCP function without affecting Wg signaling. In dsh1 hemizygous males, an average of 16.8 cells with multiple hairs (12% of the cells) are present in this region. When Rok is overexpressed via a tubP-Drok transgene in the dsh1 hemizygous background, the average number of cells exhibiting multiple hairs is reduced by more than 7-fold to 2.3 per wing region. tubP-Drok expression in a wild-type genetic background does not give rise to any obvious phenotypes. Suppression of the dsh1 multiple hair phenotype by tubP-Drok expression could also be seen when F-actin-based prehairs were visualized in the pupal wing. In contrast to the suppression of the dsh1 phenotype by overexpression of Rok, reduction of rok dosage by 50% (assuming that Drok2 is null) results in a 2.5-fold increase in the number of multiple hair cells (Winter, 2001).

Genetic interactions between rok and fz were examined in a different assay. The proper level of Fz/Dsh signaling is critical for the generation of wild-type PCP, since both overexpression and loss of function of these genes result in polarity defects in the eye and the wing. Overexpression of Fz 30 hr after puparium formation (APF) produces primarily a multiple hair phenotype that is suppressed by dsh1 heterozygosity. Similarly, reducing the wild-type copy number of RhoA and rok by half suppresses the phenotype by 2- to 2.5-fold. Taken together, these experiments suggest that Rok functions downstream of Fz/Dsh in restricting the number of F-actin-based prehairs (Winter, 2001).

Rok signaling regulates the phosphorylation of nonmuscle myosin regulatory light chain (MRLC), and hence the activity of myosin II. Does the phosphorylation state of MRLC modify the multiple hair phenotype of dishevelled mutants? Use was made of a series of mutant spaghetti squash (sqh) transgenes (sqh codes for the Drosophila MRLC) with point mutations in the primary (Ser-21) and secondary (Thr-20) phosphorylation sites, changing them either to glutamic acid (phosphomimetic), or to nonphosphorylatable alanine. Can the phosphorylation state of MRLC also modulate Fz/Dsh signaling? An examination was made to determine whether the phosphomimetic and nonphosphorylatable forms of MRLC could directly modify the dsh1 multiple hair phenotype. Introducing one copy of sqhE20E21 reduces the number of multiple hair cells in dsh1 mutants by 5-fold. sqhE21, or sqhA20E21, also suppresses the dsh1 phenotype by more than 2-fold. In contrast, introduction of sqhA21 into the dsh1 background enhances the multiple hair phenotype. The involvement of MRLC in the Fz/Dsh pathway was also examined using the Fz-overexpression assay. Reducing the wild-type sqh gene dosage from two to one, by introducing a single copy of the sqhAX3 null allele, results in a 2-fold suppression of the multiple hair phenotype caused by Fz overexpression. These results support the notion that MRLC functions in the PCP pathway to restrict F-actin bundle assembly to a single site (Winter, 2001).

Unlike other characterized PCP mutants that affect both orientation and number of wing hairs, the primary defect in Drok2 clones appears to be the presence of multiple hairs per cell, with little or no wing hair orientation defect. This suggested that Rok and what lies downstream are involved in transmitting a subset of the Fz/Dsh signal. Supporting this idea, it was found that tubP-Drok and sqhE20E21 suppress the multiple hair phenotype of dsh1, but not the hair misorientation phenotype. Additional data supporting this conclusion comes from observing the site of prehair initiation. Prehairs emerge aberrantly from the center of dsh1 mutant cells, rather than from the distal vertex as seen in wild type cells. Such mispositioning of prehair initiation correlates with the failure to acquire the proper distal orientation. While tubP-Drok expression suppresses multiple prehair formation, it does not affect the site of F-actin initiation in dsh1. Finally, the hair orientation defect resulting from Fz overexpression (via hs-fz) at 24 hours is suppressed by reducing dsh gene dosage but not that of RhoA, rok, sqh or ck. Taken together, these observations suggest that separate mechanisms allow Fz/Dsh to independently regulate the number and the orientation of prehairs, and that only the former involves Rok signaling (Winter, 2001).

Frizzled and Dishevelled function in determining cell polarity in the developing wing

The frizzled gene of Drosophila encodes a transmembrane receptor molecule required for cell polarity decisions in the adult cuticle. In the wing, a single trichome is produced by each cell, which normally points distally. In the absence of frizzled function, the trichomes no longer point uniformly distalward. During cell polarization, the Frizzled receptor (visualized using Frizzled-Green fluorescent protein) is localized to the distal cell edge, probably resulting in asymmetric Frizzled activity across the axis of the cell. Furthermore, Frizzled localization correlates with subsequent trichome polarity, suggesting that it may be an instructive cue in the determination of cell polarity. This differential receptor distribution may represent a novel mechanism for amplifying small differences in signaling activity across the axis of a cell (Strutt, 2001).

To understand the asymmetric distribution of Fz-GFP, the distribution was studied in flies mutant for other genes involved in trichome polarity establishment. In clones of cells lacking starry night (stan) function, both the apical and PD localization of Fz-GFP is completely abolished. However, in cells lacking dsh function, in which Fz signal transduction is compromised, Fz-GFP apical localization is preserved, but there is no proximodistal (PD) localization, with a splotchy irregular distribution being seen instead. The same phenotype is observed for mutations in the prickle-spiny-legs (pkpk-sple) and Van Gogh (Vang) genes. This would be consistent with the trichome polarity phenotypes of these mutations being due to a failure of Fz localization (Strutt, 2001).

Genetic data indicate that the polarity genes in, fy, and mwh act downstream of Fz/Dsh, inhibiting trichome formation where Fz is not active. In agreement with this, mutations in these loci do not alter Fz-GFP distribution despite trichome polarity being disrupted (Strutt, 2001).

The localization of Stan has also been reported to be disrupted in cells lacking dsh function but not in those lacking mwh. Therefore, whether Fz-GFP and Stan remain colocalized in different mutant backgrounds was tested. In clones of cells lacking dsh function, it was found that both Fz-GFP and Stan remain predominantly apical, and although the distribution of both appears diffuse, they nevertheless show broad colocalization. Similarly, in an in background, both Fz-GFP and Stan remain apical and colocalized to the PD cell boundaries (Strutt, 2001).

The following model is put forward for Fz function in the polarization of single cells in the developing wing. Initially, unlocalized Fz is required for the long-range propagation of a polarity signal. Fz is then recruited apically in a Stan-dependent manner and becomes stably localized at the distal cell edge in a process requiring Fz signaling and the activities of Stan, Dsh, Pkpk-sple, and Vang. This Fz localization then restricts the site of trichome initiation to the distal cell vertex. It is possible that Fz signaling activates Stan molecules to bind both to Fz (in the same cell) and to Stan molecules in the adjacent cell, and so anchors Fz at the distal edge of the cell. Localization of Fz may lead to further increased Fz signaling (possibly through the effects of receptor clustering), which could, in turn, recruit more Stan and Fz. Over time, increased activity of clustered Fz receptors at the distal cell edge would lead to the majority of the Fz in the cell being recruited to this location. In heterologous systems, Fz activity leads to recruitment of Dsh to the cell membrane, so it is likely that Dsh is also present at the distal cell boundary. A precedent for Fz-dependent localization of a cytoplasmic protein during planar polarity establishment is provided by the observation that the Numb protein requires Fz activity for correct asymmetric subcellular localization during sense organ precursor cell divisions (Strutt, 2001).

The stable PD localization of Fz also requires Pk-Sple and Vang activity, with their loss having a similar effect on Fz-GFP localization as loss of Dsh activity. It is possible that, like Dsh, they are required for the transduction of the Fz signal, or they may be involved in the function of Dsh itself. Interestingly, Vang activity on only one side of the PD boundary is sufficient for Fz-GFP localization to occur. Further investigations of the biochemical activities of these proteins will be required to fully elucidate their roles in planar polarity establishment (Strutt, 2001).

Dishevelled and the frizzled pathway

The Drosophila epidermis is characterized by a dramatic planar or tissue polarity. The frizzled pathway has been shown to be a key regulator of planar polarity for hairs on the wing, ommatidia in the eye, and sensory bristles on the notum. The genetic relationships between putative frizzled pathway downstream genes inturned, fuzzy, and multiple wing hairs (inturned-like genes) and upstream genes such as frizzled, prickle, and starry night (frizzled-like genes) were investigated. Previous data has shown that the inturned-like genes are epistatic to (function downstream of) the frizzled-like genes when the entire wing is mutant. Consistent with this are observations showing that the asymmetric accumulation of Fz, Dsh, and Fmi is not altered in in, fy, or mwh mutants. Those experiments were extended and the behavior of frizzled clones in mutant wings was examined. The domineering nonautonomy of frizzled clones is not altered when the clone cells are simultaneously mutant for inturned, multiple wing hairs, or dishevelled but it is blocked when the entire wing is mutant for inturned, fuzzy, multiple wing hairs, or dishevelled. Thus, for the domineering nonautonomy phenotype of frizzled, both inturned and multiple wing hairs are needed in the responding cells but not in the clone itself. Expressing a number of frizzled pathway genes in a gradient across part of the wing repolarizes wing cells in that region. inturned, fuzzy, and multiple wing hairs are required for a gradient of frizzled, starry night, prickle, or spiny-legs expression to repolarize wing cells. These data argue that inturned, fuzzy, and multiple wing hairs are downstream components of the frizzled pathway. To further probe the relationship between the frizzled-like and inturned-like genes the consequences of altering the activity of frizzled-like genes was determined in wings that carried weak alleles of inturned or fuzzy. Interestingly, both increasing and decreasing the activity of frizzled and other upstream genes enhances the phenotypes of hypomorphic inturned and fuzzy mutants. Results from several different experimental paradigms support the hypotheses that in and fy function downstream of fz, dsh, stan, pk, and sple in the wing and that they are required for the transduction of the fz signal to regulate hair morphogenesis and hence the actin and microtubule cytoskeletons (Lee, 2002).

To determine the relative positions of in, fy, and mwh with respect to several other fz-like tissue polarity genes, their ability to block the gain-of-function phenotypes that result from the directed expression of stan/fmi, pk, and sple were examined. ptc-GAL4 was used to overexpress pk. This results in a band of expression in the C region of the wing, with a decreasing gradient of expression away from the center of the ptc expression domain. This results in hairs pointing toward the midline in the C region of the wing. This phenotype is blocked by mutations in in, fy, and mwh. The resulting wings (e.g., ptc-GAL4/UAS-pk; in/in) show no effects from the directed expression of pk. Two different GAL4 drivers (ms1096-GAL4 and act-GAL4) were used to direct the expression of UAS-sple. Both of these drivers result in relatively even expression across the wing (although in ms1096 the level in the dorsal cell layer is higher than that in the ventral cell layer). This results in a reversal of hair polarity over much of the wing that resembles that seen in pkD. Once again it was found that mutations in in, fy, and mwh are able to block this gain-of-function phenotype. The omb-GAL4 driver was used to drive expression of UAS-stan. In the wing disc omb-GAL4 drives expression in a band located centrally along the anterior/posterior axis of the wing. However, in the pupal wing the expression pattern is more complicated during the time for tissue polarity development. During this time in the distal region of the wing, a series of alternating bands of expression and no expression is seen. Driving expression of stan using omb-GAL4 leads to a series of bands of polarity reversals; polarity is oriented from cells of low toward cells of high stan expression. Mutations in in, fy, and mwh are able to block the consequences of overexpressing stan. These data argue that in, fy, and mwh functioned downstream of fz, pk, sple, and stan in tissue polarity (Lee, 2002).

Driving expression of dsh using the ptc-GAL4 driver results in the formation of multiple hair cells in the proximal part of the ptc expression domain. In addition, ectopic bristles are also often found, presumably due to the role of dsh in canonical wg signaling. When UAS-dsh +/+ ptc-GAL4; in/in flies were examined, an additive multiple hair cell phenotype was found (i.e., stronger than either single phenotype) in the proximal part of the ptc domain and the presence of ectopic bristles. Equivalent results were found in UAS-dsh fy; fy ptc-GAL4 wings. The formation of the ectopic bristles was not surprising, since this is likely an effect of dsh on canonical wg signaling and in and fy are not thought to play a role in wg signaling. The additive multiple hair cell phenotype was unexpected and argues either that in and fy are not downstream of dsh in the fz pathway or that when overexpressed, Dsh can bypass the requirement for in and fy function (Lee, 2002).

The overexpression of fz just prior to hair initiation results in a multiple hair cell phenotype, which resembles that of in and fy (this is sometimes referred to as the late fz gain of function). When fz was overexpressed just prior to hair initiation in pupae that were mutant for a null allele of either in or fy an increased number of multiple hair cells was found. Thus, for the late fz gain of function, as for the overexpression of dsh, either in and fy are not required for fz signal transduction or when fz is overexpressed late the requirement for in and fy can be bypassed (Lee, 2002).

To test whether the frizzled-like genes could be regulating the inturned-like genes negatively or positively, the effects of both decreased and increased fz-like gene activity on hypomorphic alleles of in and fy were examined. In these experiments advantage was taken of the much higher number of multiple hair cells found in in and fy mutants compared to fz-like mutants. In all cases examined, mutations (null or hypomorphic) in fz, pk, Vang, stan, and dsh acted as strong enhancers of a weak in or fy phenotype as assayed by the frequency of multiple hair cells. These data are consistent with the fz-like genes acting as positive regulators of in and fy (Lee, 2002).

If there were a simple positive relationship between the fz-like genes and in and fy then it would be expected that the overexpression of fz-like genes should suppress hypomorphic alleles of in and fy. To test this fz was overexpressed from a hs-fz transgene and it was found that this enhances the multiple hair cell phenotypes of weak inII53 and fyJN12 alleles. These results argued that fz antagonizes the activity of in and fy. The genetic relationship between pk, sple, and fmi and weak alleles of in and fy was similarly tested. Overexpression of sple and stan from ms1096-GAL4 (or act-GAL4); UAS-sple and omb-GAL4; UAS-stan, respectively, in hypomorphic mutant backgrounds of in and fy results in an increase in the multiple hair cell phenotypes in the region of the wing, where expression is driven by the GAL4 enhancer trap. A similar enhancement was not seen for the overexpression of pk from ptc-GAL4 UAS-pk. These results suggest that sple and stan antagonize the activity of the in and fy. Notably, the gain-of-function polarity phenotypes of overexpressed pk, sple, and stan are blocked even with weak alleles of in and fy, confirming that these genes are required for the function of the fz pathway. It is clear that the fz-like genes do not act as simple positive or negative regulators of in and fy. The interaction is reminiscent of the observations that similar phenotypes result from either overexpression or a lack of function of fz-like genes (Lee, 2002).

The disruption of the microtubule cytoskeleton by treatment with vinblastine or colchicine results in many wing cells forming more than one hair. At the level of the individual cell these multiple hair cells resemble those found in in and fy mutants since the hairs are formed at the cell periphery and appear to be the result of independent initiation events. The altered polarity seen in in and fy mutant cells is not induced by vinblastine or colchicine. Immunostaining has not revealed any defects in microtubule organization in in or fy mutants. Thus, it seems unlikely that the in and fy phenotypes are due to disrupting the microtubule cytoskeleton and in this way indirectly affecting hair morphogenesis. However, it seemed possible that the disruption of the microtubule cytoskeleton could be inducing multiple hairs by interfering with in and/or fy function. It was reasoned that if that is the case then treatment of in or fy null mutant wing cells with vinblastine or colchicine would not produce a stronger phenotype than the in or fy null cells by themselves. Vinblastine was injected into in or fy mutant pupae and an additive response was found. That is, injected in or fy mutant cells produces a stronger multiple hair cell phenotype than does untreated mutant cells or cells from injected wild-type pupae. This result shows that the microtubule disruption phenotype cannot be due to a simple interference with in/fy function and argues that the microtubule cytoskeleton has a function that is independent of the fz pathway. The additive response in this experiment stands in sharp contrast with the lack of additivity in double mutants of in, fy, frtz, and mwh (Lee, 2002).

The hypothesis that in, fy, and mwh function downstream of fz and dsh originally came from the observation that mutations in in, fy, and mwh are epistatic to mutations in fz and dsh. Consistent with this hypothesis, the distal accumulation of Fz, Dsh, Fmi, and Dgo is not altered in in, fy, or mwh mutants. in, fy, and mwh are required for cells to either detect or respond to the lack of fz activity in a clone of neighboring mutant cells. The function of in and fy was required for cells to respond to a directed gradient of fz, stan, pk, or sple expression. Thus, using both of these experimental approaches it was found that in and fy are required for the function of the fz pathway. The simplest interpretation of this is that in and fy are essential downstream components of the fz pathway in the wing. An alternative explanation is that in and fy function in a parallel pathway and that the function of the fz pathway is dependent on this putative in pathway. There were a couple of surprising exceptional results. The overexpression of dsh or the late overexpression of fz induces the formation of multiple hair cells and it was found that these phenotypes are additive with an in or fy mutation. This could indicate that there are multiple pathways downstream of fz and dsh in the wing and that one of them does not require in or fy function. It is thought more likely that this is an example of overexpression bypassing a normal requirement for downstream proteins. It is suggested that In and Fy normally function as adapters to allow distally accumulated Fz and Dsh to stimulate the cytoskeleton and induce hair initiation in the correct part of the cell. When Fz or Dsh is overexpressed just prior to hair initiation the high cellular concentration might allow these proteins to bypass the need for In and Fy and to interact with and hyperactivate the hair initiation machinery (Lee, 2002).

In vertebrate embryos, a fz-based planar polarity pathway regulates convergent extension. Thus far there has been no evidence for a fz-based planar pathway having an analogous role in Drosophila. The eversion defect seen in fz mutant wings may be such an example. Some fz flies have a deformed wing that is short, fat, and often kinked proximally. This is seen in all strong and null fz alleles examined. This is due to a defect in wing disc eversion. In a normal pupa the wing everts so that it extends posteriorly and ventrally. In a fz mutant many pupae contain wings that extend anteriorly and sometimes dorsally. The wing eversion phenotype is another phenotype where in and fy appear to be epistatic to fz. The observations are somewhat puzzling as the defect in fz appears to be rescued by a mutation in in or fy. Thus, it is not clear that a simple inactivation of the fz pathway is responsible for the eversion phenotype. Perhaps for this phenotype there are multiple inputs into the fz pathway upstream of in and fy and the eversion defect is due to an imbalance in the input to in and fy. Blocking the pathway at in or fy would eliminate the function of the pathway completely and suppress miseversion (Lee, 2002).

The cellular mechanisms involved in the eversion of the wing are only poorly understood, but it is possible that convergent extension plays a role. It is also possible that the joint defects seen in fz mutants have a similar basis. In vivo observations on everting wings and joint morphogenesis would make an important contribution toward determining if convergent extension plays a role in these morphogenetic events. The development of in vivo imaging approaches for Drosophila pupae makes such experiments possible (Lee, 2002).

Wnt4, along with frizzled2 and dishevelled, regulates the dorsoventral specificity of retinal projections in the Drosophila melanogaster visual system

In Drosophila, the axons of retinal photoreceptor cells extend to the first optic ganglion, the lamina, forming a topographic representation. DWnt4, a secreted protein of the Wnt family, is the ventral cue for the lamina. In DWnt4 mutants, ventral retinal axons misproject to the dorsal lamina. DWnt4 is normally expressed in the ventral half of the developing lamina and DWnt4 protein is detected along ventral retinal axons. Dfrizzled2 and dishevelled, respectively, encode a receptor and a signaling molecule required for Wnt signaling. Mutations in both genes caused DWnt4-like defects, and both genes are autonomously required in the retina, suggesting a direct role of DWnt4 in retinal axon guidance. In contrast, iroquois homeobox genes are the dorsal cues for the retina. Dorsal axons accumulate DWnt4 and misproject to the ventral lamina in iroquois mutants; the phenotype is suppressed in iroquois:Dfrizzled2 double mutants, suggesting that iroquois may attenuate the competence of Dfrizzled2 to respond to DWnt4 (Sato, 2005).

Frizzled family receptors and Dishevelled are required for a wide variety of Wnt signaling cascades7. Dfz2 and dsh mutant flies have ovarian defects similar to those of DWnt4, strongly suggesting that Dfz2 and Dsh are involved in DWnt4 signaling. A ventral-to-dorsal misrouting phenotype was observed in Dfz2 and dsh mutant backgrounds. The results suggest that Dfz2 and Dsh are involved in DWnt4 signaling for R axon guidance. In addition, the greater expressivity and penetrance of Dfz2 and dsh mutants suggests the involvement of other Wnt family ligands in this process. However, after examining mRNA expression of all the known D. melanogaster Wnt genes, no such genes were found acting as ventral cues in concert with DWnt4. DWnt2 is expressed just outside the lamina, but its expression is symmetric along the dorsoventral axis (Sato, 2005).

The accumulation of DWnt4 on the surface of ventral R axons implies reception of the ligand and subsequent signal activation in R cells. Consistently, Dfz2 is localized on the surface of the anterior-most R axons, but not on surrounding lamina cells. To test whether Wnt signaling autonomously regulates axon guidance, dsh homozygous clones were induced in the retina using ey-Gal4:UAS-flp18. Surprisingly, the axonal misrouting phenotype was rarely observed despite the presence of many dsh mutant clones in the retina. When the retina was entirely dsh homozygous, the same dsh allele showed axonal misrouting. It is suspected that mutant R axons project normally in the presence of surrounding wild-type axons due to axon fasciculation. If this is the case, R axons homozygous for dsh may show a misrouting phenotype in the absence of neighboring, wild-type R cells. To test this idea, GMR-hid was introduced in trans to the dsh mutant chromosome. Wild-type R cells eventually die by programmed cell death triggered by hid expression behind the furrow. In this context, a severe misrouting phenotype was observed that was ventral-to-dorsal. Notably, DWnt4 protein accumulation was observed along ventral axons that were mutant for dsh and had misprojected to the dorsal lamina. Retina-specific Dfz2 clones with GMR-hid also showing a ventral-to-dorsal misrouting phenotype. These observations are consistent with the idea that DWnt4 expressed in the lamina directly regulates R axon projections (Sato, 2005).

There are two Wnt signaling pathways: the canonical and noncanonical pathways. In the former, ß-catenin/Armadillo (Arm) and TCF/Pangolin (Pan) form a complex to activate target gene transcription. In the latter, Wnt signaling is transduced independently of Arm and Pan. Canonical Wnt signaling was manipulated using UAS-panN, which encodes a constitutive repressor form of Pan, and UAS-arm, which encodes a constitutively active form of Arm. Misrouting along the dorsoventral axis was hardly observed in either genotype. Thus it is concluded that canonical Wnt signaling plays a very minor role, if any, in dorsoventral specification of R axon guidance. The above results strongly suggest the involvement of noncanonical Wnt signaling in R cells. To confirm this idea, UAS-dshDEP, which acts as a dominant-negative mutant in noncanonical signaling, was expressed in the retina. Again a strong ventral-to-dorsal phenotype was observed (Sato, 2005).

Although the planar cell polarity (PCP) pathway is categorized as a noncanonical Wnt pathway transduced by the Fz receptor, no PCP defects were observed in DWnt4 and Dfz2 mutant retinae. In addition, the retinotopic phenotype was not observed in fz null mutant backgrounds. These results suggest that DWnt4 regulates R axon projections via a noncanonical Wnt signaling distinct from the PCP pathway. wingless (wg) is involved in the specification of the dorsal retina through the activation of iro expression. The distinct chiral forms of ommatidia in the dorsal and ventral retina reflect the dorsoventral specification of the retina and the PCP signaling. The normal iro expression and the normal ommatidial chirality suggest that axonal misroutings occur independently of the retinal dorsoventral specification in DWnt4 and Dfz2 backgrounds. Since dsh is required for PCP signaling and the specification of the dorsal retina, ommatidial chirality was disorganized and dorsal iro expression was eliminated in dsh retinae. However, the expression of Serrate (Ser), which is specific to the ventral retina in wild-type backgrounds, was not affected, suggesting that the ventral cell fate is correctly specified in dsh homozygotes. Additionally, UAS-dsh and UAS-dshDEP expression under the control of GMR-Gal4 did not affect the dorsoventral specification of the retina as visualized by iro and Ser expression. Note that GMR-Gal4 is expressed behind the morphogenetic furrow well after the dorsoventral specification at earlier stages. The data shown above suggest that dsh also regulates R axon projections independently of the dorsoventral patterning of the retina (Sato, 2005).

Wnt/PCP proteins regulate stereotyped axon branch extension in Drosophila

Branching morphology is a hallmark feature of axons and dendrites and is essential for neuronal connectivity. To understand how this develops, the stereotyped pattern of Drosophila mushroom body (MB) neurons, which have single axons branches that extend dorsally and medially, was analyzed. Components of the Wnt/Planar Cell Polarity (PCP) pathway were found to control MB axon branching. frizzled mutant animals showed a predominant loss of dorsal branch extension, whereas strabismus (also known as Van Gogh) mutants preferentially lost medial branches. Further results suggest that Frizzled and Strabismus act independently. Nonetheless, branching fates are determined by complex Wnt/PCP interactions, including interactions with Dishevelled and Prickle that function in a context-dependent manner. Branching decisions are MB-autonomous but non-cell-autonomous as mutant and non-mutant neurons regulate these decisions collectively. Wnt/PCP components do not need to be asymmetrically localized to distinct branches to execute branching functions. However, Prickle axonal localization depends on Frizzled and Strabismus (Ng, 2012).

Branching morphogenesis is a key feature in axons and dendrites. This study shows that Wnt/PCP proteins, known for their roles in tissue polarity, are involved in two aspects of axon branch formation: stereotyped extension and collective decision-making. Some PCP mutants have a preference for dorsal or medial branching phenotypes. As the genetic results suggest that Fz and Stbm functions are separable, this is distinct from some tissue polarity models that have been presented for Drosophila patterning. The results suggest that PCP proteins have complex cross-regulatory effects that include the spatial regulation of Pk. A balance of their functions is key to stereotyped branching and to axon growth and guidance (Ng, 2012).

An independent study recently reported similar results for Wnt/PCP components in MB axon branching, guidance and growth (Shimizu, 2011). When comparing the results with those described in this study, most of the data are consistent. Slight differences, such as the immunohistochemistry results and the role of Prickle, might be a result of differences in preparative methods, stages at which animals were analyzed and the genetic background of the animals used (Ng, 2012).

Branched axons and dendrites tend to follow a bifurcation motif. In MB axons, this corresponds to a single dorsal and a single medial projection. This study shows that PCP proteins control the extension of axon branches, rather than the initial branch formation. The working model is that growing axons can initially reach the axon peduncle and bifurcate independently of the Wnt/PCP pathway. Once the nascent branches have formed, Fz and Stbm direct the stereotyped extension of branches. Fz is the major determinant of dorsal projections. By contrast, Stbm predominantly controls medial projections. The results suggest that these are partly redundant systems; dorsal and medial projections also require minor contributions from Stbm and Fz, respectively. In the absence of both, branching errors are increased and randomized. In some cases, both extensions are lost. Axon branching relies mainly on mutual cooperation between Fz and Stbm. In some cases, antagonism between Stbm and Fz exists, although there is no evidence of Stbm inhibiting Fz signals. These results are reminiscent of observations in Drosophila showing that Fz and Stbm can exhibit redundant and cooperative PCP functions in the wing as well as in sensory organ precursor cell fate specification (Ng, 2012).

MB axons follow a branching mode that reflects collective behavior. The initial results suggest that PCP mutant phenotypes are recessive. Next to wild-type axons, PCP mutant axons tend to adopt a wild-type branched pattern. However, with the GFPstbm rescue paradigm, the results together suggest that it is the collective mix of mutant and non-mutant neurons that determines whether an axon branches or not. How is this regulated? One possibility is that as MB axons are fasciculated, cell-cell contacts can regulate collective branching behavior. Although axons are long and thin, it is unlikely that 1000 branched αβ neurons in each hemisphere are in contact with each other. It is more likely that, as in other Drosophila tissues, it is the behavior of cells that are widely interlinked together that is controlled by PCP factors. The non-autonomous results presented in this study differ significantly from previous PCP studies. Previous results in Drosophila wing and eye show that PCP mutations result in both cell-autonomous and non-autonomous tissue polarity defects. In this study PCP branch functions are not fully cell-autonomous. Furthermore, if domineering non-autonomy exists, it does not strongly affect wild-type axon branching (Ng, 2012).

Despite the uncertainty about its role, asymmetric organization of PCP proteins is a key feature in tissue polarity. However, there are variations to this theme. For example, although Fz and Stbm are often on opposite poles, in mouse eye lens fiber cells they appear in the same tissue pole. In zebrafish and ascidian models in which PCP signals control the convergence and extension of cells in the developing notocord, PCP protein localizations are variable. These and other observations have led to the suggestion that in motile cells, PCP proteins might be dynamically regulated. In short, despite PCP mutants having different preferences in dorsal or medial branch defects, no obvious PCP protein asymmetry can be found between axon branches. One possibility is that these proteins are regulated dynamically, therefore such differences are not readily observable in fixed tissue. Another possibility is that distinct branching fates might arise from differences in PCP effector functions between branches (Ng, 2012).

The observation that Pk axonal branch levels are reduced in fz and stbm mutants suggests that Pk regulation forms part of PCP branch functions. This is likely to be post-translational and axonal-specific as Pk localization in MB dendrites is unaffected by fz or stbm. This is similar to Drosophila wing patterning for which Fz loss also results in a cell-autonomous reduction of Pk. However, in the wing, Dsh, Fz and Stbm also regulate the intercellular accumulation of Pk at the cell cortex, along the pole vertices. Also, in contrast to some wing models, this study shows that Fz and Stbm branch functions can still occur independently of Pk (Ng, 2012).

Several results suggest that PCP proteins function early during axonal development. First, Pk expression is higher in the αβ core region, where newborn developing MB axons are located. Further evidence from the stbm rescue studies shows that GFPstbm expression in the αβ core alone is sufficient to rescue axon branching. Also, branching defects were apparent in PCP mutants at early stages of development. Lastly, Pk and Dsh αβ localization can also be detected at these stages (Ng, 2012).

How Wnt/PCP proteins interact has been studied extensively in Drosophila. This study shows that the MB axon phenotypes reveal some novel features of PCP signaling interactions. As already mentioned, Fz and Stbm play partly redundant, parallel roles. Although Pk controls axon branching and is regulated by Fz and Stbm, it does not account for all Fz and Stbm functions. This is also evident from other PCP studies. However, Pk does act additively to Fz and Stbm and has convergent as well as antagonistic functions with Dsh. Also, this study has shown that Dsh does not simply transduce Fz or Stbm signals. This was also reported in some studies. The working model is that both Fz and Stbm are likely to function in part through Dsh and Pk. Based on further experiments, their effects are likely to be complex and context-dependent on the activity levels of other PCP proteins (Ng, 2012).

Previous reports suggest that these factors interact physically with each other. There is evidence that the extracellular regions of Stbm and Fz can physically bind to each other, although this is debated. Physical associations between Stbm-Dsh and Fz-Dsh have been documented in vertebrate and fly proteins. Apart from Stbm, Pk also interacts physically with Dsh. Recent reports suggest that mouse Stbm (Vangl2) and Dsh (Dvl1) interact antagonistically to regulate Frizzled3 (Fzd3) phosphorylation. Whether these interactions affect axon branching is currently unknown. Nonetheless, novel Wnt/PCP interactions are involved in MB axon patterning (Ng, 2012).

In conclusion, this study demonstrates that Wnt/PCP proteins are key regulators in the stereotyped growth of axon branches. Given that branched growth is intrinsic to neuronal morphogenesis, Wnt/PCP components and their reported interactions might also be involved synapse and dendrite formation. Such highly cross-regulated programs are probably essential for the intricate patterning of the nervous system (Ng, 2012).

Trimeric G protein-dependent Frizzled signaling in Drosophila: G proteins act upstream of Dsh, Sgg, and Arm

Frizzled (Fz) proteins are serpentine receptors that transduce critical cellular signals during development. Serpentine receptors usually signal to downstream effectors through an associated trimeric G protein complex. However, clear evidence for the role of trimeric G protein complexes for the Fz family of receptors has hitherto been lacking. This study documents roles for the Galphao subunit (Go) in mediating the two distinct pathways transduced by Fz receptors in Drosophila: the Wnt and planar polarity pathways. Go is required for transduction of both pathways, and epistasis experiments suggest that it is an immediate transducer of Fz. While overexpression effects of the wild-type form are receptor dependent, the activated form (Go-GTP) can signal when the receptor is removed. Thus, Go is likely part of a trimeric G protein complex that directly tranduces Fz signals from the membrane to downstream components (Katanaev, 2005).

The evidence that Go transduces Wg signaling comes from the analysis of Go mutants, from overexpression studies, and from the epistasis experiments. These are addressed in the following discussion (Katanaev, 2005).

Further evidence for the role of Go in transducing Wg comes from the overexpression experiments. When Go is overexpressed in the wing disc, clear upregulation of Wg targets is evident. If Go achieves the upregulation of the target genes by hyperactivating the intracellular Wg transduction machinery, then abrogation of transduction downstream of Go should nullify its effects. To this end, it was shown that the upregulation of Wg targets is arm and dsh dependent and is abolished by overexpression of sgg. Furthermore, Go overexpression in embryos gives gain-of-function wg phenotypes that are arm dependent (Katanaev, 2005).

In arm and dsh clones (and fz, fz2 clones described below), residual Dll expression was sometimes found. This occurs in otherwise wild-type tissues and in both anterior and posterior domains of hh-Gal4; UAS-Go wing discs and is most noticeable with dsh known for strong perdurance. However, arm and dsh clones in the regions of Go overexpression lose Dll expression to a level comparable with clones in which Go is not overexpressed. Thus, it is inferred that the upregulation of Wg targets induced by overexpression of Go requires the Wg transduction pathway utilizing Dsh, Sgg, and Arm (Katanaev, 2005).

The epistasis experiments provide two key indications that Go represents an immediate transducer of Fz signaling. (1) Dsh (previously the highest element of the transduction cascade identified downstream of the receptors) is necessary for the effects of Go overexpression. (2) Since serpentine receptors act as exchange factors for trimeric G proteins, the effects of overexpression of a wild-type form should require the presence of the exchange factor to load and subsequently reload GTP. Conversely, once loaded with GTP, the form lacking GTPase activity (Go-GTP) will be a long-lived activated subunit. Thus, if Fz acts as the exchange factor for Go, then it would be expected that wild-type Go would require Fz for its overexpression effects but that the activated form would be significantly less dependent. This is what was observed: Wg signaling is significantly rescued in fz, fz2 cells concomitantly expressing Go-GTP as compared to those expressing wild-type Go (Katanaev, 2005).

Given that Go functions in the Wg transduction pathway, given that its overexpression effects require Dsh, and given that its activated form is receptor independent, the simplest explanation is that Go functions in a trimeric G protein complex that relays signals from Fz receptors. These data do not necessarily suggest that Go is the exclusive transducer of Wg signals: other trimeric complexes may be involved, and non-G protein-mediated signaling may also occur (Katanaev, 2005).

In the wing, the key molecular events associated with PCP occur by 30 hr APF, when Fz becomes specifically localized to the distal membrane of the cell. The localization of Fz appears to require its own signaling, since, in dsh mutants, Fz localization does not occur. A similar effect occurs when Fz is overexpressed: Fz is no longer restricted to the distal membrane. Given this complexity, the following feature of Go can be predicted if it indeed acts as a transducer of Fz signaling. (1) Loss of Go activity should induce PCP phenotypes; (2) Fz localization should not occur correctly when Go signaling is compromised. In regard to these two predictions, it has been shown that (1) reduction of Go function or Go overexpression induces clear PCP defects and (2) Fz localization is aberrant when Go function is down- or up-regulated. Furthermore, it has been shown that Go itself undergoes a striking asymmetric redistribution in a fz-dependent manner (Katanaev, 2005).

A mutational analysis of dishevelled in Drosophila defines novel domains in the dishevelled protein as well as novel suppressing alleles of axin

Drosophila dishevelled (dsh) functions in two pathways: it is necessary to transduce Wingless (Wg) signaling and it is required in planar cell polarity. To learn more about how Dsh can discriminate between these functions, genetic screens were performed to isolate additional dsh alleles and the potential role of protein phosphorylation was examined by site-directed mutagenesis. Two alleles were identified with point mutations in the Dsh DEP (Dishevelled, Egl-10, Pleckstrin) domain that specifically disrupt planar polarity signaling. When positioned in the structure of the DEP domain, these mutations are located close to each other and to a previously identified planar polarity mutation. In addition to the requirement for the DEP domain, it was found that a cluster of potential phosphorylation sites in a binding domain for the protein kinase PAR-1 is also essential for planar polarity signaling. To identify regions of dsh that are necessary for Wg signaling, a screen was carried out for mutations that modified a GMR-GAL4;UAS-dsh overexpression phenotype in the eye. Many alleles of the transgene containing missense mutations were recovered, including mutations in the DIX (Dishevelled, Axin) domain and in the DEP domain, the latter group mapping separately from the planar polarity mutations. In addition, several transgenes had mutations within a domain containing a consensus sequence for an SH3-binding protein. Second-site-suppressing mutations were recovered in axin, mapping at a region that may specifically interact with overexpressed Dsh (Penton, 2002).

By using three independent experimental approaches, a set of new mutations has been identified in Dsh that disrupt two distinct signaling events. These new mutations map to specific regions within Dsh and thus provide important information on the function of the different protein domains. A screen was carried out for mutations in the endogenous dsh gene that disrupt planar polarity and a misexpression phenotype was utilized to screen for mutations that affect Wg signaling. Mutations that disrupt the Wg signaling function of Dsh occur throughout the protein while mutations that disrupt planar polarity signaling are confined to the DEP domain. However, the mutation of potential phosphorylation sites positioned between the basic region and the PDZ domain of Dsh (cluster ST4) specifically disrupts the ability of Dsh transgenes to rescue the tissue polarity phenotype. The mutations uncovered in this work are discussed by domains, beginning at the N terminus (Penton, 2002).

DIX domain mutations: Seven UAS-dsh alleles encode missense mutations that map to the DIX domain and all reduce or abrogate Wg signaling in three separate assays. These results clearly demonstrate that the DIX domain is required for Wg signaling and agree with other studies in Drosophila. Furthermore, studies of the mammalian homolog of Dsh (Dvl1) show that the DIX domain of Dvl can interact directly with itself and that it binds to axin. The N-terminal DIX domain in Dsh shares 37% amino acid identity with the C-terminal DIX domain of Axin. The Axin DIX domain also interacts with itself and is necessary to regulate the stability of ß-catenin in SW48O cells. This indicates that DIX domains are important for protein-protein interactions and that the DIX domain of Dsh may mediate Wg signaling by binding to and inhibiting Axin (Penton, 2002).

The basic region: Dsh possesses a highly conserved basic region (aa 219–228) of unknown function. No point mutations were isolated in this region in the genetic screens. Moreover, deletion of the basic region compromises neither the function of Dsh in Wg signaling nor its function in planar polarity signaling. It is noted, however, that two of the transgenic lines carrying the DshDeltabasic construct under control of the hsp70 heat-shock promoter rescue the lethality of the dshv26 null allele even in the absence of heat shock, in contrast to all other lines tested. This result points to a potential role of the basic region as a negative regulator of the signaling function of Dsh (Penton, 2002).

PDZ domain mutations: Surprisingly, only one allele, UAS-dsh8-1, was recovered that maps to the PDZ domain. It only mildly attenuates the Dsh eye misexpression phenotype and does not attenuate the Dsh wing misexpression phenotype at all. While this result might suggest that there is only a minor requirement for this domain in Wg signaling, it could also mean that single point mutations have little effect on the function of the PDZ domain. Deletion of the PDZ domain completely abolishes Dsh function in both Wg signaling and tissue polarity. This result contrasts with other studies which show that the PDZ domain is not necessary to rescue the embryonic lethality of dsh null embryos. This discrepancy may be due to the fact that different types of rescue assays were used to study the domain requirements of Dsh. Heat-shock-inducible transgenes were used in this study that allowed complete rescue to adulthood under nearly physiological conditions, whereas a previous study overexpressed different mutant versions of Dsh by RNA injection into embryos. The construct used in these experiments deletes amino acids 287–336, which lie within the PDZ domain, while the construct that was previously used removes amino acids 152–333, consisting of the basic domain, CK1–CK4 and ST1–ST5. Thus an alternative explanation is that the previously utilized construct deletes a region that inhibits the activity of Dsh. This is consistent with the observations implicating the basic domain as a negative regulator of Dsh activity. Other studies have found a requirement for the PDZ domain in regulating ß-catenin stability and in regulating transcription of LEF reporter constructs. In addition, the PDZ domain binds to Axin, FRAT, CK1, CK2, PP2A, and IDAX, all proteins that regulate Wg signaling. Thus, while the PDZ domain may be dispensable for the function of Dsh under certain experimental conditions, it appears to be essential for Wg signaling and tissue polarity under physiological conditions (Penton, 2002).

SH3-binding domain mutations: Three new UAS-dsh alleles were isolated that carry mutations in a novel domain of Dsh. This region lies between the PDZ domain and the DEP domain, is proline rich, and possesses a consensus sequence for a class I core SH3 protein-binding motif, RTEPVRP at position 352–358. Proline-rich sequences that contain this core domain mediate the binding of these proteins to SH3 proteins. This core motif is conserved in the mammalian homologs of dsh and so are the surrounding prolines. UAS-dsh8-12 mutates the last proline in this core binding motif into a leucine at position 358, and UAS-dsh8-79 and UAS-dsh8-68 mutate an aspartic acid at position 360 to valine and a glycine at position 362 to aspartic acid, respectively. The proline and aspartic acid residues are conserved between dsh and its dvl homologs, while the glycine residue is replaced by an alanine residue in the dvl genes. UAS-dsh8-12 and UAS-dsh8-79 disrupt but do not completely abolish Wg signaling while UAS-dsh8-68 possesses more Wg signaling activity than does either UAS-dsh8-12 or UAS-dsh8-79. This could be because UAS-dsh8-68 maps further from the core SH3-binding site motif or because the amino acid that it mutates is not conserved. Although the mutations encoded by UAS-dsh8-79 and UAS-dsh8-68 do not map within the core SH3-binding consensus motif, it is known that amino acids that surround this motif are important for optimal ligand preference. Studies utilizing combinatorial peptide libraries have defined binding sequences for SH3 proteins and show that proline-rich sequences around the core domain are important for binding (Penton, 2002).

The identification of mutations in a putative SH3-binding domain is intriguing since these proteins have not been implicated in Wg signaling events. Interestingly, the cytoplasmic tails of Arrow and DFz2 also contain putative SH3-binding domains. In addition, D-Axin contains putative SH3-binding domains in the RGS domain, the ß-catenin-binding domain, and immediately amino terminal to the DIX domain. SH3 proteins act as adaptors linking signaling molecules into complexes and localizing proteins to the cell membrane. Hence it is tempting to speculate that proteins with multiple SH3 domains participate in Wg signaling events, perhaps to localize Dsh and Axin in proximity to the Arrow and DFz2 cell surface receptors. Indeed, Dsh is localized to the cell membrane when it is coexpressed with Fz1 in Xenopus oocytes. It has also been suggested that the DEP domain is important for this localization during planar polarity signaling (Penton, 2002).

DEP domain mutations: The isolation of two planar polarity alleles that encode mutations within the DEP domain agrees with other studies that demonstrate a requirement for this domain in planar polarity signaling. The dshA3 mutation maps four amino acids distal to the previously isolated dsh1 allele and encodes an arginine-to-histidine mutation at position 413. The dshA3 and dsh1 mutations replace a positively charged amino acid with a neutral amino acid, suggesting that Dsh may contact negatively charged proteins during planar polarity signaling or bind to membrane phospholipids. dshA21 encodes a cysteine-to-arginine mutation at position 472. Cysteine residues can be palmitoylated and such a lipid modification can be important for cell membrane attachment (although attempts were made to incorporate labeled palmitate in Dsh and none was detected). Thus it is possible that this mutation disrupts the ability of the DEP domain to become membrane localized during planar polarity signaling. On the structure of the DEP domain, the dshA3 and dsh1 mutations are located fairly close to the cysteine that is mutated in dshA21, suggesting that these three residues are collectively involved in the function of the DEP domain (Penton, 2002).

Despite the fact that the DEP domain is viewed to be specific for polarity signaling, three new UAS-dsh alleles were obtained that disrupted Wg signaling. These mutations cluster to a region of the DEP domain extending from position 440 to 459, away from the dsh planar polarity alleles. This indicates that the DEP domain is required for Wg signaling, in agreement with findings that dsh constructs that lack the DEP domain cannot rescue the embryonic lethality of a dsh null allele (Penton, 2002).

Although all UAS-dsh alleles that were tested either lost or attenuated the Wg signaling function, nearly all caused planar polarity defects when expressed in the wing. Both gain and loss of signaling activity alter planar cell polarity. Therefore, it is hard to determine whether these alleles contain mutations that are specific for Wg signaling and that leave planar polarity functions intact, or if they act as dominant negatives for this function. Another possibility is that the Wg signaling function of dsh is more sensitive to perturbations in dsh activity than is the planar polarity function and that these alleles behave as hypomorphs. Indeed lower levels of a dsh transgene are required to rescue planar polarity functions of dsh than to rescue Wg signaling functions. Three dsh alleles exist that specifically perturb planar polarity, however, arguing that planar polarity functions and Wg signaling functions are separable. In addition, when constructs that contain the dsh1 mutation are overexpressed they cannot rescue the endogenous dsh1 mutation, arguing that dsh1 is not a hypomorph and that Dsh acts as a modular protein (Penton, 2002).

Phosphorylation mutants: Dsh is a phosphoprotein and Wg signaling generates hyperphosphorylated forms of Dsh, which are enriched in membrane fractions in biochemical assays. While this finding suggests that the phosphorylation state of Dsh may regulate its activity, no mutations were identified in putative phosphorylation sites in the forward screens. Furthermore, in vitro mutagenesis of putative phosphorylation sites of Dsh in a region spanning amino acids 178–254 do not reveal a requirement for these sites in the Wg signaling function of Dsh. Thus, phosphorylation sites in Dsh may be redundant and more than one site may need to be mutated to produce a phenotype. This conclusion is supported by phosphotryptic mapping experiments, which identified at least three phosphorylation sites in Dsh (Penton, 2002).

Surprisingly, however, region ST4 is essential for planar polarity signaling. This region binds the protein kinase PAR-1, which is thought to act in Wg signaling rather than in tissue polarity. It is noted, however, that the PAR-1 kinase is implicated in generating cell asymmetry in Caenorhabditis elegans and in the Drosophila oocyte. These results point to a potential role of PAR-1-mediated Dsh phosphorylation in planar polarity (Penton, 2002).

Second-site suppressors -- mutations in axin: In addition to mutations in the UAS-Dsh transgene itself, the UAS-dsh misexpression screen yielded second-site modifiers on the third and fourth chromosome. Modifiers on the first and second chromosome could not be recovered due to the strategy of the screen. Five of the second-site modifiers map near axin and were indeed found to contain mutations within the axin gene. They behave as dominant suppressors of Dsh misexpression phenotypes in both the wing and eye but do not modify Wg or DFz2 misexpression phenotypes. In addition, these alleles are homozygous viable and have no phenotype when they are recombined away from UAS-dsh. Since Axin normally suppresses Wg signaling, and null axin alleles do not interact with UAS-dsh, it is inferred that these alleles specifically suppress overexpressed forms of Dsh but do not affect Dsh that is regulated by Wg signaling. This would imply that overexpressed Dsh works through a mechanism that is different from Dsh when activated by Wg. For example, overexpressed Dsh may interact with Axin through binding to a domain that is different from the Axin domain that interacts with Wg-activated Dsh (Penton, 2002).

Conclusions: This work and other studies suggest that Dsh is a modular protein with specific domains dedicated to Wg and planar polarity signaling. How is Dsh activity regulated and how does it mediate Wg signaling? The targeting of Dsh to the cell membrane and the regulation of its phosphorylation state are correlated with Wg and planar polarity signaling. The DEP domain is necessary to localize Dsh to the cell membrane and a putative SH3-binding site has been identified that may also be important for membrane localization of Dsh. It will be interesting to examine the phosphorylation state of the Dsh protein that is produced from the alleles generated in these screens and to determine if protein from alleles that mutate the potential SH3-binding site are membrane localized. CK1, CK2, and PAR-1 bind to Dsh and phosphorylate it. Moreover, CK1 and PAR-1 are positive regulators of Wg signaling that promote stabilization of ß-catenin and induce the expression of Wnt target genes. Thus, Dsh localization to the cell membrane may be controlled by CK1 and PAR-1; this localization may lead to changes in Dsh activity (Penton, 2002).

Antagonistic interactions between widerborst and dishevelled

widerborst (wdb), a B' regulatory subunit of Drosophila PP2A (Microtubule star), is a conserved component of planar cell polarization machinery in Drosophila; the zebrafish homolog also functions in planar cell polarity mechanisms. In Drosophila, wdb acts at two steps during planar polarization of wing epithelial cells. It is required to organize tissue polarity proteins into proximal and distal cortical domains, thus determining wing hair orientation. It is also needed to generate the polarized membrane outgrowth that becomes the wing hair. Widerborst activates the catalytic subunit of PP2A and localizes to the distal side of a planar microtubule web that lies at the level of apical cell junctions. This suggests that polarized PP2A activation along the planar microtubule web is important for planar polarization. In zebrafish, two wdb homologs are required for convergent extension during gastrulation, supporting the conjecture that Drosophila planar cell polarization and vertebrate gastrulation movements are regulated by similar mechanisms (Hannus, 2002).

widerborst, in German, means something stubborn or recalcitrant (derived from wider, meaning against, and borst, meaning bristle). It encodes a B' regulatory subunit of protein phosphatase 2A (PP2A), an enzyme that is conserved from yeast to mammals. PP2A is a holoenzyme that consists of a catalytic (C) subunit, an A regulatory subunit and one of a large family of B, B' or B'' subunits. The latter subunits are thought to regulate the activity of the C subunit and provide substrate specificity. In metazoans, the B' subunits have diverged into two related subclasses. The central regions of these proteins are strongly conserved, but they differ at their N and C termini. The protein encoded by widerborst is more closely related to the human alpha, ß and epsilon subunits (62%-66% identity) than to the ß or gamma subunits (52%-59% identity). Its sequence suggests that wdb might influence tissue polarization by regulating PP2A activity with respect to specific targets (Hannus, 2002).

To ask whether cortical polarization might depend on Widerborst, the localization of Flamingo and Dishevelled was examined in cells expressing dominant-negative Wdb along the AP compartment boundary. The cells that express dnWdb are identifiable by their elevated staining with Widerborst antibody. In these wings, it is clear that expression of dnWdb causes both Fmi and Dsh to accumulate uniformly around the cortex at high levels. By contrast, Dsh and Fmi are normally polarized in most of the cells that do not express dominant negative Wdb. These data indicate that Wdb activity is required for normal cortical polarity (Hannus, 2002).

Interestingly, the expression of dnWdb appears to inhibit the distal accumulation of endogenous wild type Wdb in the cells adjacent to the dnWdb-expressing domain. This observation is consistent with the non-autonomous effects on hair formation observed in the adult wings of these flies. Furthermore, Dsh and Fmi are often depolarized in cells up to five cell diameters away from the dnWdb-expression domain. This might either be due directly to lower levels of endogenous Widerborst, or to non-autonomous propagation of cortical depolarization in the dnWdb-expression domain (Hannus, 2002).

To determine if Widerborst is required for the cytoskeletal integrity of wing epithelial cells, the effect of dominant negative expression on actin, microtubules and Coracle was examined. Cortical organization of both filamentous actin and Coracle appears essentially normal in dnWdb-expressing cells, suggesting that failure to polarize Flamingo and Dishevelled distribution does not result from gross defects in the subcortical actin cytoskeleton. By contrast, the organization of the planar microtubule web is perturbed by expression of dominant negative Wdb. Although dnWdb-expressing cells accumulate microtubules to at least normal levels, their ordered, web-like structure is not maintained. This suggests that Wdb normally directs the dephosphorylation of a protein that is important for microtubule organization and that the structure of the planar web may contribute to the development of cortical polarity (Hannus, 2002).

Widerborst is needed for convergent extension movements during zebrafish gastrulation. The PP2AB' regulatory subunits of the alpha/epsilon family are highly conserved and homologous genes are present from worms to humans. To ask whether the function of widerborst has been conserved in different cellular processes that require cell polarization, whether it plays a role in the regulation of gastrulation movements in zebrafish was examined. One of the main cellular rearrangements during gastrulation in zebrafish and Xenopus is convergent extension. In convergent extension, cells move to the dorsal side of the gastrula to redistribute there along the forming anteroposterior body axis. Convergent extension movements are driven by mediolateral cell intercalations that require prior mediolateral polarization of cells. In recent studies, it has been shown that convergent extension movements depend on some of the same proteins that are responsible for organizing planar polarity in Drosophila, including zebrafish homologs of dishevelled and strabismus/Van Gogh. Two zebrafish widerborst homologs (wdb1 and wdb2) have been identified, that both clearly fall into the alpha/epsilon subclass of B' regulatory subunits; the proteins they encode are 63% and 64% identical to Dm and Wdb, respectively. Zebrafish wdb1 and wdb2 are maternally provided and expressed during all stages of gastrulation. To address their role(s) in convergent extension, wdb1 and wdb2 morpholino antisense oligonucleotides were injected alone and in combination into one-cell-stage embryos and the resulting phenotypes were examined at bud stage. Embryos injected with low concentrations of wdb1 and wdb2 morpholinos exhibit a shortened and broadened body axis at the end of gastrulation as monitored by in situ hybridization using notail (notochord), dlx3 (anterior edge of neural plate) and hgg (prochordal plate) as markers. This phenotype suggests that the embryos are defective in convergent extension movements. Embryos injected with higher concentrations of the same morpholinos were strongly dorsalized as seen by an expanded domain of gsc expression in the presumptive shield anlage at the onset of gastrulation (Hannus, 2002).

These data suggest that zebrafish Wdb regulates dorsoventral axis formation, as well as convergent extension, but that convergent extension is more sensitive to the dose of Wdb. For both phenotypes, injections of zebrafish wdb1 and wdb2 morpholinos alone causes similar although sometimes weaker phenotypes than injections of a combination of both. These data may suggest that, although Wdb1 and Wdb2 perform the same functions, sufficient protein levels are only attained when both genes are expressed. Alternatively, they may have non-redundant functions in both dorsoventral axis formation and gastrulation (Hannus, 2002).

Since the Drosophila and zebrafish Widerborst proteins show a high degree of sequence conservation, whether injection of the Drosophila dominant-negative Widerborst and PP2A-C subunit constructs interfers with convergent extension movements was tested in zebrafish. Injections of both dominant-negative constructs cause reduced convergent extension movements while dorsoventral patterning is largely unaffected, indicating that the Drosophila PP2A B' alpha/epsilon subunit fulfills similar functions in zebrafish and Drosophila (Hannus, 2002).

How might Wdb operate to specify cortical polarity? When Wdb activity is reduced, components of the cortical domains like Dsh and Fmi accumulate uniformly around the cell cortex at high levels. By contrast, disruption of Frizzled signaling interferes with the accumulation of Dsh and Fmi at the cell cortex. This suggests that Wdb is not required to activate Frizzled signaling, but rather is important for making it asymmetric (Hannus, 2002).

The genetic data indicate that Wdb exerts its activity by activating the catalytic subunit of PP2A with respect to specific substrates, and the localization of Wdb suggests that it does so on the distal side of the planar microtubule web. Which proteins might be targeted for dephosphorylation by Widerborst? One possibility is Dishevelled. Heterozygosity for wdb strongly suppresses the mwh phenotype of dsh1, suggesting that during tissue polarization these two proteins act antagonistically. Dishevelled cortical localization correlates with hyperphosphorylation, and the cortical localization of Dsh is certainly expanded in Wdb dominant-negative expressing cells. Supporting this possibility, two-hybrid experiments have indicated that Dishevelled can physically interact with a Xenopus B' regulatory subunit. If Wdb normally acts by antagonizing Dsh, then the dominant-negative might overactivate Frizzled signaling and cause defects in tissue polarity. This model is not easily reconcilable with a role for the distal localization of Wdb; one might naively expect an antagonist of Frizzled signaling to accumulate proximally instead of distally. Nevertheless, although the early distal localization of Wdb is suggestive, it has not been proven relevant to cortical polarization; for example, Wdb might have a role in transducing the Frizzled signal, for which distal localization is not required (Hannus, 2002).

What might be the importance of Wdb binding to the distal microtubule web? Binding to the cytoskeleton might simply allow stable distal localization of an otherwise diffusible cytosolic molecule. More interesting, this association raises the possibility that Widerborst directs the dephosphorylation of a microtubule-associated protein. Consistent with this idea, the structure of the planar microtubule web is disrupted by dnWdb expression. PP2A activity is important for the accumulation of stable microtubules. Microtubule stability can affect the binding of microtubule motor proteins and can contribute to polarized protein delivery. In the wing, microtubules have been suggested to play important roles in hair polarity: depending on the time at which it is added, vinblastine treatment of pupal wings causes either failure of hair outgrowth or the formation of multiple wing hairs. Polarized dephosphorylation of MAPs within the planar microtubule web might bias the transport of vesicles containing components of the proximodistal cortical domains. At later stages, it might also help direct transport of components of the hair formation machinery to the distal side of the cell, or promote the stability of microtubules in the outgrowing hair. This model for Widerborst action could provide a single explanation for its effects on hair outgrowth and on cortical polarity. Identification of the relevant Widerborst substrate(s) should greatly advance understanding of the cell biology of tissue polarization (Hannus, 2002).

These data support other studies indicating that B' alpha/epsilon regulatory subunits antagonize the classical Wnt signaling pathway. Experiments in Xenopus embryos and tissue culture cells have shown that increasing the level of a B' alpha subunit inhibits Wnt signaling and causes ventralization. Consistent with this, experiments show that reducing Wdb levels causes dorsalization of zebrafish embryos. Although Wdb, like Frizzled and Dishevelled, is a shared component of both planar polarization and classical Wnt signaling pathways, it probably has different functions in each; during classical Wnt signaling, the B' alpha is thought to act downstream of Dishevelled, forming part of a ß-catenin degradation complex that plays no role in planar polarity signaling (Hannus, 2002).

The observation that widerborst is needed both for distal polarization of Drosophila wing hairs and for convergent extension movements during zebrafish gastrulation points to a conserved role for Wdb in regulating tissue polarity in development. Furthermore, it provides additional evidence supporting the conjecture that components of the planar polarization pathway in Drosophila are also used to control cell polarity and movement during vertebrate gastrulation. To date, the evidence for this is based on analysis of various dsh constructs and, more recently, on the analysis of vang/stbm and rhoA during vertebrate gastrulation. The identification of Wdb as another shared component provides further evidence that this signaling cascade is indeed conserved between Drosophila and vertebrates. Additional experiments will have to address the precise function(s) of vertebrate wdb homologs and where wdb acts in the genetic pathway regulating vertebrate gastrulation movements (Hannus, 2002).

Dishevelled and dorsal closure

At the end of germband retraction, the dorsal epidermis of the Drosophila embryo exhibits a discontinuity that is covered by the amnioserosa. The process of dorsal closure (DC) involves a coordinated set of cell-shape changes within the epidermis and the amnioserosa that result in epidermal continuity. Polarization of the dorsal-most epidermal (DME) cells in the plane of the epithelium is an important aspect of DC. The DME cells of embryos mutant for wingless or dishevelled exhibit polarization defects and fail to close properly. The role of the Wingless signalling pathway in the polarization of the DME cells and DC was investigated. The ß-catenin-dependent Wingless signalling pathway is required for polarization of the DME cells. Although the DME cells are polarized in the plane of the epithelium and present polarized localization of proteins associated with the process of planar cell polarity (PCP) in the wing, e.g., Flamingo, PCP Wingless signalling is not involved in DC (Morel, 2004).

The initiation of DC in Drosophila embryos correlates with the elongation and polarization of the DME cells in the DV axis of the embryo. In parallel with this polarization, a cable of F-actin assembles on the dorsal-most surface of these cells and promotes the formation of filopodia and lamellae during the final phases of the process. A functional link between the polarization and the assembly of the cable of actin is supported by the observations that in mutants in which the DME cells do not elongate, there is no actin cable and no dynamic protrusions. As a consequence, these embryos display defects and delays in the closure process. Embryos mutant for wg and dsh are good examples of this class (Morel, 2004).

The polarization of the DME cells occurs in the plane of the epithelium and can be seen as a manifestation of the phenomenon of planar cell polarity (PCP). Since a specific branch of Wg signalling has been implicated in PCP and there is evidence for an interaction between Dishevelled and JNK signalling during dorsal closure, whether there is a role for this mode of Wg signalling in the process of DC was tested. The results clearly show that the 'canonical' Wg signalling pathway that leads to activation of Armadillo and of the transcription of target genes is necessary and sufficient to restore the polarity of the DME cells and to promote a normal process of dorsal closure in a wg mutant embryo. Surprisingly, it was found that the PCP pathway does not appear to play a major role in DC or the polarization of the DME cells, since activation of the 'canonical' pathway in the absence of dsh activity rescues the polarity and function of the DME cells.

Dsh contains three highly conserved domains, the DIX, PDZ and DEP domains. The DEP domain mediates interaction of Dsh with the cell cortex and is required for PCP but not 'canonical' Wg signalling, while the DIX domain is required for the 'canonical' Wg signalling but seems dispensable for PCP. To investigate an involvement of the PCP pathway in the activities of the DME cells during DC, rescue experiments of wg embryos were carried out using truncated forms of Dsh deleted for either the DEP (DshDeltaDEP) or the DIX (DshDeltaDIX) domain. Although overexpression of DshDeltaDEP leads to the partial rescue of naked cuticle and of En expression, neither naked cuticle nor rescue of En expression are observed in wg>da>DshDeltaDIX (using da-GAL4 to drive DshDeltaDIX in wg mutants) embryos. This thus confirms that DshDeltaDEP is able to signal within the 'canonical' Wg pathway but not DshDeltaDIX (Morel, 2004).

Then the ability of either protein to rescue DC in wg embryos was tested. wg>da>DshDeltaDEP embryos are longer than wg mutants and their dorsal cuticle is improved; no hole is observed and only occasional warts can be seen. The DME cells are oriented in the DV direction and most of them show a slight elongation in the DV direction when the zippering process has started. Simultaneously, Fmi is observed at the membrane and accumulates at the level of the ANCs. Although no clear elongation of DME or ventral epidermal cells is observed, DC process is improved; two zippers, at the anterior and posterior ends of the embryo, are initiated, whereas only the posterior one is observed in wg embryos. By contrast, wg>da>DshDeltaDIX embryos have a shorter cuticle than wg mutants and show a more severe puckering and hole on the dorsal side. Furthermore, neither the shape nor the polarization of DME cells is improved in these embryos (Morel, 2004).

Thus, although DshDeltaDEP can rescue partially the DC defects of wg mutants, ubiquitous overexpression of DshDeltaDIX does not rescue any of the observed features confirming the requirement for the Wg 'canonical' pathway during DC. Thus, the conclusion that the PCP pathway does not appear to play a role in DC or in the polarization of the DME is supported by the observation that although a moiety of Dishevelled that promotes Armadillo signalling is capable of rescuing the defects of wg mutants, a moiety that promotes JNK signalling and PCP does not. Altogether, these results indicate that the polarization and activity of the DME cells during dorsal closure requires Armadillo/ß-catenin-dependent Wg signalling. Furthermore, this requirement is restricted to the epidermis because activation of Wg signalling in the amnioserosa has no effect on the epidermis (Morel, 2004).

The notion of PCP has emerged from studies of the mechanism that determines the orientation of the hairs in the cells of the wing of Drosophila. A number of studies have revealed the existence of protein complexes that mediate this orientation by becoming asymmetrically distributed between the proximal and distal membranes of the epidermal cells. Thus, while Flamingo becomes localised equally between the proximal and the distal sides of the cell, the distal side of the cell accumulates a complex composed of Frizzled and Dishevelled and the proximal side accumulates a complex formed by Strabismus and Prickled. Genetic analysis of these complexes has led to the formulation of a model which describes the propagation of the polarity from one cell to its neighbours, but which says nothing about the origin of the polarity that is being propagated. In this model, Dsh, like Strabismus, Prickled or Frizzled, is an essential component of the mechanism that propagates the polarity (Morel, 2004).

The observation of polarized distributions of Fmi, Dsh and Fz in the DME cells during dorsal closure has led to the suggestion of a link between the polarization of these cells and the process of PCP. However, no requirement has been found for elements of this pathway in dorsal closure. In particular, the PCP function of Dsh is not required for the polarization of the DME cells and the polarized localization of Fmi, which was quite unexpected considering the interdependence of Dsh and Fmi for their asymmetric localization in the wing. This asymmetric distribution of Fmi is likely to play a role in the polarized actin dynamics in response to the polarity signal. Although this may appear surprising, it also invites a consideration of the notion of PCP (Morel, 2004).

The PCP pathway has been defined in a context of propagation of a polarity but not of its initial definition. In fact none of the experiments performed in the wing of Drosophila address the origin of the polarity that is being propagated. In DC, however, the process that was observed in the asymmetric distribution of proteins in the DME cells reflects the establishment of a polarity and not its propagation. From this perspective, the lack of a requirement for the PCP branch of Wnt signalling might not be that surprising as PCP Wnt signalling might be related to propagation or coordination of a polarity signal that has been generated in a different manner. However, the requirement for the ß-catenin-dependent Wg pathway might be significant and indicate the requirement for a transcriptional event in the establishment of PCP. This observation might also apply to the wing (Morel, 2004).

Dishevelled and Oogenesis

Identifying the signals involved in maintaining stem cells is critical to understanding stem cell biology and to using stem cells in future regenerative medicine. In the Drosophila ovary, Hedgehog is the only known signal for maintaining somatic stem cells (SSCs). Wingless (Wg) signaling is also essential for SSC maintenance in the Drosophila ovary. Wg is expressed in terminal filament and cap cells, a few cells away from SSCs. Downregulation of Wg signaling in SSCs through removal of positive regulators of Wg signaling, dishevelled and armadillo, results in rapid SSC loss. Constitutive Wg signaling in SSCs through the removal of its negative regulators, Axin and shaggy, also causes SSC loss. Also, constitutive wg signaling causes over-proliferation and abnormal differentiation of somatic follicle cells. This work demonstrates that wg signaling regulates SSC maintenance and that its constitutive signaling influences follicle cell proliferation and differentiation. In mammals, constitutive ß-catenin causes over-proliferation and abnormal differentiation of skin cells, resulting in skin cancer formation. Possibly, mechanisms regulating proliferation and differentiation of epithelial cells, including epithelial stem cells, are conserved from Drosophila to man (Song, 2003).

Wg produced from terminal filament and cap cells may reach SSCs at a distance of a few cells by either diffusion or active transport, and then Wg directly controls SSC maintenance. Furthermore, correct intermediate levels of wg signaling seem to be important for maintaining SSCs in the Drosophila ovary. Reduction of wg signaling in SSCs by removal of positive regulators such as arm and dsh causes rapid SSC loss, as does constitutive wg signaling in SSCs by removal of negative regulators such as Axn and sgg. wg signaling maintains SSCs through several possible mechanisms: (1) wg signaling could be required for SSC self-renewal and/or survival; (2) it could maintain the association of SSCs with IGS cells, and/or (3) both mechanisms could work simultaneously. DE-cadherin-mediated cell adhesion has been shown to be important for keeping SSCs in their niche; it also shares arm as a common component with wg signaling. wg signaling is known to regulate levels of arm, which are also important for DE-cadherin-mediated cell adhesion. Thus, it is possible that wg signaling regulates cell adhesion between SSCs and their niches. In addition, arm mutant clonal analysis strongly argues that wg signaling must also directly regulate SSC self-renewal and/or survival. arm2 mutant SSC clones are lost very quickly over time in comparison with wild-type SSC clones, and the arm2 mutation primarily affects wg signaling but does not disrupt DE-cadherin-mediated cell adhesion. Therefore, wg signaling controls SSC maintenance through regulating SSC self-renewal/survival and/or cell adhesion between SSCs and their niche cells. The temperature-sensitive allele of wg gives very mild phenotypes in follicle cell production, however, removal of wg downstream components has a dramatic impact on SSC maintenance. In Drosophila, there are six other wg-related genes. This raises an interesting possibility that other wg-like molecules could also be involved in regulating SSC maintenance (Song, 2003).

CKIε/discs overgrown promotes both Wnt-Fz/β-catenin and Fz/PCP signaling in Drosophila

The related Wnt-Frizzled(Fz)/β-catenin and Fz/planar cell polarity (PCP) pathways are essential for the regulation of numerous developmental processes and are deregulated in many human diseases. Both pathways require members of the Dishevelled (Dsh or Dvl) family of cytoplasmic factors for signal transduction downstream of the Fz receptors. Dsh family members have been studied extensively, but their activation and regulation remains largely unknown. In particular, very little is known about how Dsh differentially signals to the two pathways. Recent work in cell culture has suggested that phosphorylation of Dsh by Casein Kinase I ε may act as a molecular 'switch', promoting Wnt/β-catenin while inhibiting Fz/PCP signaling (Cong, 2004). This study demonstrates in vivo in Drosophila through a series of loss-of-function and coexpression assays that CKIε acts positively for signaling in both pathways, rather than as a switch. The data suggest that the kinase activity of CKIε is required for peak levels of Wnt/β-catenin signaling. In contrast, CKIε is a mandatory signaling factor in the Fz/PCP pathway, possibly through a kinase-independent mechanism. Furthermore, the primary kinase target residue of CKIε on Dsh has been identified. Thus, the data suggest that CKIε modulates Wnt/β-catenin and Fz/PCP signaling pathways via kinase-dependent and -independent mechanisms (Klein, 2006).

Cell-culture assays have suggested that CKIε positively regulates Wnt-Fz/β-catenin signaling and that it antagonizes Fz/PCP signaling. To confirm that CKIε is required for Wnt/β-catenin signaling in vivo, loss of function (LOF) alleles of discs overgrown (dco/doubletime, the Drosophila CKIε gene) were examined for phenotypes indicative of Wingless signaling defects. Consistent with previous data demonstrating a requirement for dco in disc growth, strong dco/CKIε alleles (dcodbt-P) gave clones too small to analyze for disruption of Wg target-gene expression. Thus dco/CKIε mutant clones were generated with the Minute technique. In these, expression of the Wg target gene senseless (sens) was lost in mutant cells. Accordingly, dco/CKIε adult wing clones show loss of margin bristles and/or parts of the wing margin, consistent with a positive requirement for dco in Wg signaling. However, Wg targets that require lower levels of Wg signaling (e.g., Dll) were not affected, indicating that dco/CKIε is only required for peak Wg signaling levels. Consistent with this finding, genome-wide RNAi screens for Wg signaling components identified dco/CKIε as a factor required for peak levels of β-catenin reporter expression (Klein, 2006).

The Fz/PCP pathway can easily be studied in Drosophila. The precise ommatidial arrangement in the eye and the orientation of hairs on the wing depend on correct Fz/PCP input. The two best-studied PCP signaling factors are Fz and Dsh, which also act in canonical Wnt/β-catenin signaling. To study the role of dco/CKIε in PCP, various heteroallelic dco combinations were analyzed. In several of these (e.g., dcodbt-P/dcodbt-AR), typical PCP defects were seen. Clones of a strong dco/CKIε allele show classical PCP phenotypes in the wing, with reoriented wing hairs, and in the eye, with ommatidal chirality and orientation defects (dco/CKIε clones also displayed ommatidia with photoreceptor loss, likely as a result of the cell viability requirement of Dco/CKIε (Klein, 2006).

In vivo LOF analyses allow led to the conclusion that dco/CKIε is required for peak levels of Wg signaling, but does not appear to be a mandatory Wnt/β-catenin signaling component. In addition, the data identify dco/CKIε as a new factor required in Fz/PCP signaling (Klein, 2006).

To dissect the function of dco/CKIε in Wg and PCP signaling, the effects of overexpressing CKIε were examined. In the eye, dco/CKIε was overexpressed with sevenless(sev)-Gal4 (in R3/R4 cells that are critical for PCP establishment, which causes PCP phenotypes. In the wing, decapentaplegic(dpp)-Gal4-driven expression in a proximal-distal stripe along the A-P compartment boundary can be used to identify positive and negative effects on both Wg and PCP signaling. dpp>CKIε displayed a mild but consistent PCP defect, a characteristic hair swirl near the intersection of the dpp stripe and wing margin. It also caused a small number of extra margin bristles, typical of increased Wg signaling (Klein, 2006).

Given that these phenotypes were mild, attempts were made to enhance them. Because CKIε requires an activating dephosphorylation event, which can be induced by Fz signaling, the effect was tested of coexpressing CKIε with either Fz or Fz2 in the dpp stripe. Overexpression of Fz (dpp>Fz) causes a characteristic reorientation of wing hairs that point away from the expression domain, but does not induce ectopic margin bristles. Strikingly, coexpression of Fz and CKIε leads to a dramatic synergy, with enhanced PCP defects and a large number of extra margin bristles in the expression domain, indicative of a positive CKIε role in both Wg and PCP signaling. Consistently, expression of Fz or CKIε alone is not sufficient to induce visible changes in Wg target-gene expression, but coexpression of CKIε and Fz cell-autonomously induces ectopic Senseless-positive cells (Klein, 2006).

dpp>Fz2 induces many ectopic margin bristles near the intersection of dpp expression and the wing margin (at 25°C;). Because dpp-driven coexpression of Fz2 and CKIε at 25°C is lethal, the dpp>Fz2, CKIε coexpression was examined in flies raised at 18°C (allowing for weaker Gal4-driven expression). dpp>Fz2 at 18°C induces only few margin bristles, a phenotype that is enhanced upon coexpression with CKIε; dpp>Fz2, CKIε coexpression also induces PCP-like hair swirls, although this effect could be indirect given that this combination induces wing-margin-like vein tissue, which could repolarize parts of the wing blade). Taken together with the LOF analyses, these data demonstrate a positive, synergistic role for Dco/CKIε not only in Wg signaling, but also in Fz/PCP signaling (Klein, 2006).

To confirm that dco/CKIε acts positively for both pathways in vivo, genetic interactions of dco with known Wg and PCP signaling factors were examined in the eye. Overexpression of Fz (sevFz) causes strong PCP defects, a phenotype that is significantly suppressed by the removal of a single copy of dco. Overexpression of Strabismus (Stbm; also known as Van Gogh or Vang), an antagonist of Fz/PCP signaling, with sevGal4 (sevStbm) causes mild PCP defects, which are enhanced by removal of a copy of dco, again supporting a positive role for dco in Fz/PCP signaling (Klein, 2006).

The effect of removing a copy of dco in the context of Dsh overexpression (sev>Dsh). sev>Dsh causes both PCP defects and loss of photoreceptors, with the latter resembling the effect of sev>Wg. Removing a copy of dco strongly suppresses the loss-of-photoreceptor phenotype, supporting a positive role for dco for peak Wg signaling. Assessing the effect of the removal of a copy of dco on the PCP in sev>Dsh is not possible, because the loss-of-photoreceptor phenotype masks PCP defects in many ommatidia (Klein, 2006).

In addition, genetic analyses was performed with overexpressed CKIε. sev>CKIε eyes exhibit mild PCP defects and a small percentage of ommatidia with a change in photoreceptor number. Removal of a single copy of dsh suppressed the PCP and photoreceptor number defects, consistent with Dco/CKIε acting positively together with Dsh to elicit both phenotypes. Consistent with the coexpression results in the wing, these interactions support a positive role for dco/CKIε in both Wg and Fz/PCP signaling (Klein, 2006).

On the basis of the genetic interaction data with Dsh and cell-culture and in vitro kinase assays that have shown that CKIε can bind and phosphorylate Dsh, Dsh appears to be a likely phosphorylation target of CKIε. The actual site of phosphorylation on Dsh, however, has not been mapped. To narrow down the region of phosphorylation, a series of GST-Dsh constructs were generated covering all domains. In vitro kinase assays using CKIε showed specific phosphorylation of all Dsh isoforms containing the basic region and PDZ domain (GST-Dsh, GST-bPDZ, GST-ΔC), but not of those containing just the PDZ (GST-PDZ) or other parts of Dsh (Klein, 2006).

To determine the exact site of phosphorylation, the region N terminal to the PDZ domain was analzyed for conserved CKIε consensus sites, and a likely motif was identified. In this motif, S236 is predicted to be the first serine residue phosphorylated by CKIε. When this serine is mutated to alanine (within GST-bPDZ), CKIε is no longer able to phosphorylate Dsh. Interestingly, this residue is in a short region of Dsh that is important for Dsh phosphorylation, activity, and signal specificity (Klein, 2006).

Because previous studies have suggested that CKIε kinase activity is required for its ability to transduce Wnt/β-catenin signals, a kinase-dead isoform of CKIε, Dco/CKIεD132N (which affects the ATP binding site) was tested in vivo. This mutant was unable to transduce Wg signals, but, surprisingly, it induced strong GOF PCP phenotypes in both the eye and wing. sev>CKIεD132N eyes display clean PCP phenotypes. Wings from dpp>CKIεD132N flies also display PCP defects, with wing hairs that point away from the expression domain, demonstrating a GOF Fz/PCP phenotype (Klein, 2006).

To investigate the potential for a kinase-independent role of Dco/CKIε, Fz and CKIεD132N (with dppGAL4) were coexpressed. In contrast to coexpression of Fz and wild-type CKIε, CKIεD132N did not display extra margin bristles, indicating that kinase activity is important for Wg signaling. Similarly, in contrast to coexpression of Fz2 and wild-type CKIε, dpp>Fz2, CKIεD132N (at 18°C) did not cause an increase in margin bristles as compared to dpp>Fz2 alone. At 25°C, dpp>Fz2, CKIεD132N was not lethal, as compared to dpp>Fz2 together wild-type CKIε (Klein, 2006).

In summary, these data demonstrate a requirement for the Dco/CKIε kinase activity in Wg signaling, with CKIεD132N often acting as a dominant negative, but suggest that kinase activity is not required for Dco/CKIε activity during Fz/PCP signaling (Klein, 2006).

The CKIε requirement for Dsh phosphorylation was examined. In an unbiased Drosophila S2-cell-based screen for kinases that are required for the PCP-signaling-associated Dsh phosphorylation, dco/CKIε was identified as a kinase required in this context. Strikingly, the kinase-dead CKIε isoform, CKIεD132N, promotes PCP-signaling-associated Dsh phosphorylation as much as wild-type CKIε. These data suggest that although the presence of CKIε protein is important for this phosphorylation event, its kinase activity is not required, consistent with the in vivo expression data with Dco/CKIεD132N (Klein, 2006).

A possible caveat to these data is that, rather than acting on downstream targets in a kinase-independent manner, CKIεD132N could act to titrate away factors that inhibit the endogenous CKIε. To test this, the effect of CKIεD132N on the phosphorylation of Dsh was examined in S2 cells in the absence of endogenous CKIε. These data show that endogenous CKIε is not mediating the CKIεD132N effect, and thus CKIε is likely to act in a kinase-independent manner in the PCP context (Klein, 2006).

The in vivo data support a positive requirement of dco/CKIε for peak levels of Wnt/β-catenin signaling and a strict requirement in the Fz/PCP pathway. Whereas the kinase activity of CKIε is required for Wnt/β-catenin signaling, the analysis suggests that it is not required for Fz/PCP signaling. These findings differ from the proposed inhibitory effect of CKIε on Fz/PCP signaling in cell culture. It is possible that the PCP readout in cell culture, namely activation of JNK, reflects only a subset of PCP activities of Dsh, not representing an accurate measure of overall PCP activity. Alternatively, CKIε could act as a constitutively active kinase when expressed in cell culture, whereas its activity is regulated in vivo. Thus, for Fz/PCP signaling in vivo, the primary role for CKIε may not be as an active kinase, but rather as a stabilizer of a complex that allows for PCP-specific Dsh phosphorylation. This is supported by the data that the kinase activity of CKIε is not required for Fz/PCP signaling and that kinase-dead CKIε still stimulates phosphorylation of Dsh (Klein, 2006).

CKIε phosphorylates a specific residue in Dsh, S236, in a short region known to be phosphorylated by multiple kinases and suggested to be important in the regulation of Dsh signal specificity. This supports the proposed possibility of in vivo competitive phosphorylation as a mechanism for Dsh regulation. The region upstream of the PDZ domain appears to act as a docking site for Dsh binding proteins. Differential phosphorylation of this region could alter the binding properties of Dsh. In support of this possibility, protein-protein interaction studies have identified a large number of proteins that bind to the Dsh PDZ domain. It is unlikely that all these interactions occur at the same time, and phosphorylation is a potential mechanism to regulate this. Further experiments are needed to finely map the many potential phosphorylation target residues and the corresponding kinases and demonstrate their in vivo significance (Klein, 2006).

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