frizzled

DEVELOPMENTAL BIOLOGY

Effects of Mutation or Deletion (part 1/2)

frizzled affects polarity of hairs on the thorax, wing, and leg as well as the polarity of eye ommatidia. Both loss of function and overexpression of fz mutations result in an altered location for the assembly of the F-actin containing prehairs in the pupal wing. This suggests that fz functions to modulate the structure of the cytoskeleton (Park, 1994). Different frizzled mutations behave cell autonomously and non-cell autonomously, with cell-autonomous mutations occurring in a proline residue located in the presumptive first cytoplasmic loop of the protein (Jones, 1996).

In Drosophila, two closely related serpentine receptors, Frizzled and Frizzled2 are able to act as receptors for Wingless. In addition to transducing the Wg signal, Fz (but not Fz2) is able to transduce a second, unidentified signal that mediates planar polarity. Much attention has been focused on the structure of the N-termini of the Fz-class receptors and their role in ligand binding. Experiments using techniques of high-level expression have suggested a role for the C-termini in specifying which of the two second messenger systems the receptors are able to activate. It is argued here that experiments involving high level expression of the receptors cannot be adequately interpreted. The ability of the receptors and chimeric forms when driven at moderate levels to rescue loss of function of the fz and fz2 genes has been tested. Under these conditions all receptors tested will function as Wg receptors, but only a subset show the ability to rescue the polarity pathway. The presence of this subset implies that the N terminus is necessary but not sufficient and suggests that the ability to transduce the polarity signal is widely distributed throughout the protein (Strapps, 2001).

From the rescue experiments it is inferred that all chimeras assayed behaved as functional Wg receptors but only a subset was able to rescue polarity signaling in fz mutant tissue. Comparison of the chimeras that rescue polarity signaling with those that do not suggests that the N-terminal domain of Fz is critical for the transduction of the polarity signal but that alone it is not sufficient. In addition to the N terminus, one of the two other Fz domains is required. Thus the specificity for signal transduction appears spread through the three domains of the protein. The simple way to view this is that ligand binding requires the N terminus, and that transmission of the signal to intracellular molecular machinery can be achieved by at least one of two distinct sites in the remainder of the protein. It is noted that only low levels of Fz are normally required for polarity transduction and that at least two of the constructs that failed to rescue polarity transduction resulted in strong polarity phenotypes when over-expressed in the wing (Strapps, 2001).

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 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).

Mutations in the wingless gene disrupt the patterning of embryonic segments and their adult derivatives. Wg protein has been shown in cell culture to functionally interact with Frizzled2, a receptor that is structurally related to the tissue polarity protein Frizzled (Fz). However, it has not been determined if Frizzled 2 functions in the Wg signaling pathway during fly development. Overexpression of Frizzled 2 is shown to increase Wg-dependent signaling to induce ectopic margin bristle formation in developing Drosophila wings. It is suggested that the results of overexpression reflect an independence between Fz and Frizzled 2 pathways during wild-type wing development. Overexpression of a truncated form of Frizzled 2 acts in a dominant-negative manner to block Wg signaling at the wing margin, and this block is rescued by co-expression of full-length Frizzled 2 but not full-length Fz. These results suggest that Frizzled 2 and not Fz acts in the Wg signaling pathway for wing margin development. However, a truncated form of Fz also blocks Wg signaling in embryo and wing margin development; the truncated form of Frizzled 2 affects ommatidial polarity during eye development. These observations suggest that a single dominant-negative form of Fz or Frizzled 2 can block more than one type of Wnt signaling pathway and imply that truncated proteins of the Fz family lose some aspect of signaling specificity (Zhang, 1998).

Mutations in the Van Gogh gene, shown to be allelic to strabismus, result in the altered polarity of adult Drosophila cuticular structures. The two original Vang alleles were recovered because of a dominant phenotype -- a swirl in the wing hair pattern in the C' region of the wing (this is the region that lies between the third and fourth veins proximal to the proximal cross vein). On the wing, Van Gogh mutations cause an altered polarity pattern that is typical of mutations that inactivate the frizzled signaling/signal transduction pathway. Flies homozygous for Van Gogh alleles show a tissue polarity bristle phenotype on the wing, thorax, legs and abdomen. On the abdomen, bristles point almost orthogonally to the midline instead of posteriorly. The tarsus joints are often duplicated as is typical for tissue polarity mutants. The phenotype differs from those seen previously in other polarity mutants, as the number of wing cells forming more than one hair is intermediate between that seen previously for typical frizzled-like or inturned-like mutations. Consistent with Van Gogh being involved in the function of the frizzled signaling/signal transduction pathway, Van Gogh mutations show strong interactions with mutations in frizzled and prickle. Mitotic clones of Van Gogh display domineering cell nonautonomy. In contrast to frizzled clones, Van Gogh clones alter the polarity of cells proximal (and in part anterior and posterior) but not distal to the clone. In further contrast to frizzled clones, Van Gogh clones cause neighboring wild-type hairs to point away from rather than toward the clone. This anti-frizzled type of domineering nonautonomy and the strong genetic interactions seen between frizzled and Van Gogh suggest the possibility that Van Gogh is required for the noncell autonomous function of frizzled. As a test of this possibility, frizzled clones were induced in a Van Gogh mutant background and Van Gogh clones were induced in a frizzled mutant background. In both cases the domineering nonautonomy is suppressed consistent with Van Gogh being essential for frizzled signaling (Taylor, 1998).

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. Signaling mediated by the Frizzled receptor polarizes pI along this axis, thereby specifying the orientation of the mitotic spindle and positioning the Numb crescent. The mitoses of the two cells produced by mitosis of pI are oriented parallel and orthogonal, respectively, to the division axis of pI. This difference in cell-division orientation is largely independent of the identity of the secondary precursor cells, and is regulated by Frizzled-independent mechanisms (Gho, 1998).

Recently, double-stranded RNA (dsRNA) has been found to be a potent and specific inhibitor of gene activity in the nematode Caenorhabditis elegans (Fire, 1998). The potential of dsRNA to interfere with the function of genes in Drosophila, termed RNA inhibition or RNAi) has been investigated. Injection of dsRNA into embryos resulted in potent and specific interference with gene function for several of the genes that were tested. dsRNA corresponding to four genes with previously defined functions was introduced. dsRNA potently and specifically inhibits the activities of wg, fushi tarazu (ftz), even-skipped (eve), and tramtrack (ttk). The reception mechanism of the morphogen Wingless was determined using dsRNA. Interference with the function of both the frizzled and Drosophila frizzled 2 genes produces defects in embryonic patterning that mimic the loss of wingless function. Interference with the function of either gene alone has no effect on patterning. Analysis of epistasis indicates that frizzled and Drosophila frizzled 2 act downstream of wingless and upstream of zeste-white3 in the Wingless pathway. These results demonstrate that dsRNA interference can be used to analyze many aspects of gene function (Kennerdell, 1998).

The potency and specificity of dsRNA interference on gene activity suggests that it might be a useful means to eliminate Frizzled2 activity. Although the null phenotype of Fz2 is unknown, it was reasoned that if Fz2 encodes the Wg receptor, then its mutant phenotype should resemble wg loss-of-function mutants. Larvae that lack wg activity are completely covered with denticles on the ventral cuticle, unlike wild-type larvae in which ventral cuticle is an alternating pattern of naked cuticle and denticles. When dsRNA corresponding to the wg gene is injected, the region around the site of injection exhibits a wg-like mutant phenotype, and the remainder of the embryo was wild type. Surprisingly, no animals exhibit a null phenotype despite the injection of twice as much dsRNA as for other genes. The RNAi (RNA inhibition) effect is localized, and the range of phenotypes is limited by the size of the region with ectopic denticles. When dsRNA corresponding to the 5' UTR of Fz2 is injected, no effect on denticle patterning is observed. Ectopic denticles are not observed in embryos injected with dsRNA corresponding to the 5' UTR of the fz gene. In contrast, an equimolar mixture of ds-fz and ds-Fz2 RNAs causes localized transformation of naked cuticle into denticles. The RNAi effect is limited to the site of injection even at high doses of dsRNA, and its potency is highly similar to the potency of ds-wg RNA. Denticles in the affected regions resemble those typical of the fifth row in a wild-type abdominal segment, and the denticles are oriented either toward the midline or along the anteroposterior axis with reversed polarity. These features are precisely those observed in wg mutant embryos and embryos treated with ds-wg RNA (Kennerdell, 1998).

Engrailed expression initiates normally in wg mutants but fails to be maintained. To examine whether Fz and Fz2 have a similar function, embryos were injected with ds-fz and ds-Fz2 RNAs. After further development, the embryos were stained with an anti-Engrailed antibody. Expression of engrailed os absent in lateral ectoderm within the affected region. This discontinuous loss of engrailed expression resembles loss of functional wg (Kennerdell, 1998).

The interfering activity of ds-fz and ds-Fz2 RNA mixtures could mean that the fz and Fz2 genes act redundantly, and the activities of both genes must be blocked before a phenotype is observed. Alternatively, it could reflect some other synergy between the injected RNAs. ds-Fz2 RNA was injected alone into embryos mutant for fz and was found to possess interfering activity that is comparable to the interfering activity of mixed ds-fz and ds-Fz2 RNAs. These data are most consistent with the fz and Fz2 genes acting redundantly to pattern the ventral epidermis (Kennerdell, 1998).

Experiments in cell culture have suggested that Fz2 acts as a receptor in the Wg signal transduction pathway. Do Fz and Fz2 act between Wg and the intracellular components of its signal transduction pathway? Genetic epistasis can determine the order of action of genes in a common pathway. If fz and Fz2 function downstream of wg, then interference of fz and Fz2 activities should suppress activating mutations of wg. A transgenic strain that expresses high levels of Wg in all epidermal cells causes those cells to secrete naked cuticle. This strain was used to determine whether fz and Fz2 are required for wg action. When these animals are injected with ds-fz and ds-Fz2 RNAs, the formation of ectopic naked cuticle is suppressed. The injected transgenic embryos are distinct from wild-type embryos injected with RNA and from the uninjected transgenic strain. They resembled wild-type embryos injected with dsRNA in that they have denticles alternating with naked cuticle plus some localized patches of continuous denticle lawn. However, they do not usually have the complete complement of denticles. This incomplete suppression is attributed to the fact that interference of fz and Fz2 is primarily localized to regions close to the site of injection. Nevertheless, this result provides genetic evidence for a function of fz and Fz2 downstream of wg (Kennerdell, 1998).

Transduction of a Wg signal antagonizes the Shaggy/Zw3 kinase, which functions to modulate levels of Arm. Do fz and Fz2 act between wg and shaggy, as would be predicted for the Wg receptor? Loss of shaggy activity results in all epidermal cells adopting posterior segmental fates, and mutant embryos lack ventral denticles. When shaggy mutant embryos are injected with ds-fz and ds-Fz2 RNAs, there is no change in their phenotype; they resemble shaggy embryos. The similarity of the phenotypes of shaggy with or without fz and Fz2 interference suggests that fz and Fz2 function upstream of zw3 in wg signaling (Kennerdell, 1998).

There are similarities between how Wg signals to cells in the embryonic epidermis and in wing discs. Several studies suggest that Wg acts as a morphogen in both tissues. Moreover, it utilizes the same signal transduction pathway in responding cells. However, there are significant differences between how Wg works in the two tissues. In the wing disc, Wg specifies cell fate decisions but has no apparent role in controlling planar polarity of wing cells. In the embryonic epidermis, Wg specifies cell fate decisions and controls the planar polarity of cells. This planar polarity is manifested by the orientation of denticles along the anteroposterior axis, which is disrupted in wg mutants or can be redirected by wg misexpression. There is a second major difference between wing and embryo: in the wing, DFz2 and not Fz mediates the Wg signal. Misexpression of DFz2 increases the zone of Wg responsiveness in the wing, but Fz misexpression has no effect. Null fz mutants do not perturb cell fate decisions attributable to Wg. In the embryo, both Fz and DFz2 are required to mediate the Wg signal. Inhibition of both genes is sufficient to disrupt planar polarity and epidermal cell differentiation, whereas inhibition of each gene singly has no effect. These data are also consistent with experimental results in which Fz was overexpressed in embryos (Kennerdell, 1998 and references).

How can Wg, Fz, and DFz2 generate both polarity and cell fate responses in embryos and not in wing discs? One possibility is that in embryos they directly specify cell fates and indirectly affect cell polarity. For instance, they specify the diverse pattern of denticle types that might then determine overall denticle polarity. Another possibility is that distinct domains of Wg activate different cell responses by interacting with receptors in qualitatively different ways. Access to some Wg domains might be limiting in some tissues and not others. A third possibility is that Wg ligand-receptor interactions are quantitatively different in various tissues. A fourth possibility is that intrinsic factors couple Wg-bound Frizzled proteins to a particular cell response, and these factors are differentially active in various tissues (Kennerdell, 1998 and references).

Most ligands pair with specific receptors, and each pairing remains fixed for different tissues and different developmental stages. Wg appears to be an exception to this general rule. What is the significance behind Wg's diverse signaling properties? By adding greater flexibility in the competence of cells to respond to Wg, more diverse responses to a single ligand can be generated. Competence may be modified by changing the number of potential receptors and their ability to trigger more than one transduction pathway. Another reason for this diversity might be related to the function of Wg receptors in shaping the concentration gradient of Wg in a tissue. In the wing disc, high levels of Fz2 stabilize extracellular Wg and allow it to range farther from its source than in the absence of Fz2. Thus, if more than a single Frizzled protein can stabilize Wg, the combination of multiple receptor expression patterns might determine the Wg gradient. This simple combinatorial mechanism could potentially generate a broad range of gradient curves for a single ligand (Kennerdell, 1998).

The tissue polarity genes of Drosophila are required for correct establishment of planar polarity in epidermal structures. In the eye this planar polarity 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 Dishevelled acts downstream of Fz. Mutations have been identified in the Drosophila homolog of Rho1 p21 GTPase; by phenotypic analysis 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).

In cell culture assays, Frizzled and Dfrizzled2, two members of the Frizzled family of integral membrane proteins, are able to bind Wingless and transduce the Wingless signal. To address the role of these proteins in the intact organism and to explore the question of specificity of ligand-receptor interactions in vivo, a genetic analysis of frizzled and Dfrizzled2 in the embryo has been conducted. These experiments utilize a small gamma-ray-induced deficiency that uncovers Dfrizzled2. Dfz2-deficiency homozygotes die shortly after hatching and exhibit a subtle disorganization of denticle patterning with occasional ectopic denticles in posterior compartments. These data suggest that Dfz2 and/or other genes removed by the 469-2 deficiency play a minor or largely redundant role in cuticle patterning during embryogenesis. Mutants lacking maternal frizzled and zygotic frizzled and Dfrizzled2 exhibit defects in the embryonic epidermis, CNS, heart and midgut that are indistinguishable from those observed in wingless mutants. Epidermal patterning defects in the frizzled, Dfrizzled2 double-mutant embryos can be rescued by ectopic expression of either gene. In frizzled;Dfrizzled2 double mutant embryos, ectopic production of Wingless does not detectably alter the epidermal patterning defect, but ectopic production of an activated form of Armadillo produces a naked cuticle phenotype indistinguishable from that produced by ectopic production of activated Armadillo in wild-type embryos. These experiments indicate that frizzled and Dfrizzled2 function downstream of wingless and upstream of armadillo, consistent with their proposed roles as Wingless receptors. The lack of an effect on epidermal patterning of ectopic Wingless in a frizzled;Dfrizzled2 double mutant argues against the existence of additional Wingless receptors in the embryo or a model in which Frizzled and Dfrizzled2 act simply to present the ligand to its bona fide receptor. These data lead to the conclusion that Frizzled and Dfrizzled2 function as redundant Wingless receptors in multiple embryonic tissues and that this role is accurately reflected in tissue culture experiments. The redundancy of Frizzled and Dfrizzled2 explains why Wingless receptors were not identified in earlier genetic screens for mutants defective in embryonic patterning (Bhanot, 1999).

In the wild-type epidermis, wg functions in an autocrine pathway to maintain its own expression and in a paracrine regulatory loop to maintain expression of en in adjacent cells. In the epidermis at gastrulation, when wg function is first detected, a stripe of cells in the anterior half of each parasegment expresses wg and an adjacent stripe of cells in the posterior half express en. This pattern is initiated by pair-rule and gap genes, but its maintenance requires paracrine signaling by Wg to the en expressing cells and both paracrine signaling by Hh and autocrine signaling by Wg to the wg expressing cells. Thus, in wg mutant embryos the pattern of wg and en gene expression is initiated correctly but is not maintained. In fz;Dfz2 double-mutant embryos, the En stripes begin to fade at stage 9/10 and are completely absent from the epidermis by mid stage 10, similar to wg mutants. By contrast, en expression within the CNS is maintained as it is in wg mutants. Consistent with a defect in Wg signaling, Wg expression is greatly reduced in fz;Dfz2 mutants (Bhanot, 1999).

At the end of gastrulation, wg participates in the morphogenesis of various embryonic structures. In the embryonic central nervous system, wg is expressed by row 5 neuroblasts (NBs) and its function is required to specify NBs in rows 4 and 6. Null mutants of wg show a loss or duplication of several NBs, the most extensively studied being NB-4. The NB-4 lineage gives rise to two RP2 motoneurons per segment that innervate the dorsal musculature and are missing in wg mutant embryos. RP2 neurons are marked by their expression of even-skipped (eve). Mutant embryos missing maternal fz and zygotic fz and Dfz2 or missing only zygotic Dfz2 were examined using an antibody against Eve. fz;Dfz2 double-mutant embryos show a complete loss of RP2 neurons in all hemisegments. As observed in the epidermis, the fz^R52 allele shows residual activity: in fz;Dfz2 double mutants carrying the fz^R52 allele, approximately 26% of the double-mutant embryos show Eve-positive RP2 staining in 1-3 hemisegments. Interestingly, 469-2 homozygous embryos also show a weakly penetrant RP2 phenotype. In approximately 21% of the 469-2 homozygous embryos, an RP2 neuron is either missing or misplaced in 1-3 hemisegments. It is concluded that fz and Dfz2 are largely but not entirely redundant in specifying RP2 identity (Bhanot, 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 (fz^D21/fz^K21) 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).

In the preceding experiment, Stan distribution was studied in pupal wings where all the cells had lost fz expression. To dissect how Fz-dependent intercellular communication controls Stan localization, two approaches were adopted to juxtapose cells with different fz expression levels and see how those conditions affect Stan distribution. One approach generated fz mutant clones, and the other expressed fz in a gradient fashion. Clones were made of cells homozygous for a strong fz allele, fz^R52, which produces a truncated polypeptide at a very low level. Strikingly, the fz mutant cells appear to decide where to localize Stan molecules in a neighbor-dependent manner. Along clone borders, Stan accumulates at almost all the interfaces between fz⁺ (fz^R52/+ or +/+) and fz^R52/fz^R52 cells, whether the interface is a P/D cell boundary or not. In contrast, Stan is never localized intensely at boundaries between outermost mutant cells; in other words, those outermost mutant cells always restrict the distribution of Stan to contact sites with fz⁺ cells. These results indicate that every cell has a system to monitor fz expression levels across each boundary and that if there is an imbalance across a certain boundary, the cell deposits Stan molecules preferentially at this particular cell-cell contact site (Usui, 1999).

Inner mutant cells that do not contact the clone borders display fuzzy Stan signals both at interfaces between themselves and in the cytoplasm, and the distribution of the boundary signals is not polarized. These abnormal patterns are reminiscent of those in wings of the fz null mutant. This observation of the fz clone confirms that the fz gene is necessary to concentrate Stan to cell-cell boundaries and, in addition, to bias the distribution of Stan toward P/D boundaries (Usui, 1999).

Several recessive stan alleles have been isolated on the basis of the wing hair polarity phenotype seen in a wing in an FLP/FRT based F1 screen. Thus, it is clear that the presence of wild-type neighboring cells do not rescue all of the mutant cells in a clone. Several of the tissue polarity genes display domineering nonautonomy in wing clones -- that is, the presence of mutant cells in a clone alters the development of wild-type cells that are near the clone. To see if stan clones also display domineering nonautonomy, mosaic wings were generated where stan clones were marked with the hair marker pwn. Several different alleles were tested including the putative null allele stan²⁴, the recessive lethal allele stan²¹ and the recessive viable allele stan³, and similar results were obtained. In all cases, the majority of clones behaved cell autonomously. Further, the extent of domineering nonautonomy for those clones scored as showing such nonautonomy was typically much weaker than seen with fz or Vang (Van Gogh). It is concluded from these experiments that stan principally functions cell autonomously (Chae, 1999).

For an in vivo assay for fz pathway function, the domineering non-autonomy of fz clones was used. To do this, fz^R52 strb clones were induced in stan³ wings. In a wild-type wing, more than 80% of fz clones show distal domineering non-autonomy. That is, cells distal (and in part anterior/posterior) but not proximal to the clone show altered polarity that extends to cells that do not border the clone. fz^R52 strb clones were induced in regions of stan³ wings, where the polarity was consistent enough to be able to score the clones for domineering non-autonomy. Out of 54 clones, forty two clones behave cell autonomously and only 12 clones showed evidence of domineering non-autonomy. Further, the extent of domineering non-autonomy in these 12 clones was modest. Thus, stan appears to be a suppressor of the domineering non-autonomy of fz. That there remains some fz domineering non-autonomy in stan³ wings may reflect the fact that stan³ is not a null allele. The ability of a stan mutation to suppress this fz phenotype argues that stan is downstream of fz and is required for the cell non-autonomous function of the fz pathway (Chae, 1999).

As a second in vivo assay for fz pathway function, the ability of a gradient of fz expression, with its highest point near the distal tip of the wing, was used to reverse the normal distal polarity of wing hairs. This result argues that cells can 'sense' the fz activity of neighboring cells and respond to this information. The production of a region of reversed polarity is likely to require both cell non-autonomous (e.g., a fz-dependent intercellular signal) and cell autonomous functions (e.g., transduction of the fz-dependent signal). stan³ was found to completely block the ability of a gradient of fz expression to reorganize wing hair polarity. Hence it is concluded that stan functions downstream of fz and is required either for the cells to be able to sense the fz activity of neighboring cells or to respond to this information (Chae, 1999).

The overexpression of fz just prior to prehair initiation causes the formation of large numbers of multiple hair cells that are a phenocopy of the in-like mutations. This fz gain-of-function phenotype has been used as a test to identify genes that are downstream of and required for the transduction of the fz signal. The function of the dsh gene, which is thought to function downstream of fz, is indeed required for this phenocopy. However, the function of several other tissue polarity genes, pk, ds and Vang, is not required. To determine if stan is required for the transduction of the fz signal, stan;hs-fz flies were constructed and fz expression was induced just prior to prehair initiation. The stan³ does not block the ability of fz overexpression to induce cells to form multiple hairs. Rather, it appears to slightly enhance the ability of fz overexpression to induce multiple hair cells (Chae, 1999).

The frizzled gene is required for the development of distally pointing hairs on the Drosophila wing. It has been suggested that fz is needed for the propagation of a signal along the proximal distal axis of the wing. The directional domineering non-autonomy of fz clones could be a consequence of a failure in the propagation of this signal. This hypothesis was tested in two ways. In one set of experiments the domineering non-autonomy of fz and Vang Gogh (Vang) clones was used to assess the direction of planar polarity signaling in the wing. prickle (pk) mutations alter wing hair polarity in a cell autonomous way, so pk cannot be altering a global polarity signal. However, pk mutations alter the direction of the domineering non-autonomy of fz and Vang clones, arguing that this domineering non-autonomy is not due to an alteration in a global signal. In a second series of experiments, cells in the pupal wing were ablated. A lack of cells that could be propagating a long-range signal does not alter hair polarity. It is suggested that fz and Vang clones result in altered levels of a locally acting signal and the domineering non-autonomy results from wild-type cells responding to this abnormal signal (Adler, 2000).

The directional domineering non-autonomy of fz clones in the wing was originally suggested to be due to a failure in the proximal to distal propagation or transmission of a polarity signal. This model predicted a special population of cells that serve as a source (or origin) of the signal. If this model is correct how could mutations in pk and dachsous (ds) result in an altered direction of fz domineering non-autonomy? An obvious possibility is that these mutations could change the fate of some cells so that an ectopic source of signal was produced. This hypothesis is inconsistent with the cell autonomy/non-autonomy of pk clones. Consider the possibility that pk produces a tissue polarity phenotype by causing the formation of an ectopic source of signal at a new location in the wing. If a pk clone is located in such a region, then the clone would be predicted to show domineering non-autonomy. This is infrequent in pk clones, but is not restricted to clones in one or a few regions of the wing. If a pk clone is located elsewhere, it would be expected to have no consequences for polarity. However, it was observed that cells inside of all pk clones show altered hair polarity. The source and directional transmission model also fails to explain the results of temperature shift experiments with a cold-sensitive fz allele. If fz function is required for the propagation of a signal along the proximal distal axis of the wing it is predicted that a temperature shift from the permissive to the restrictive temperature during the middle of the temperature sensitive period would result in a wing with a permissive phenotype proximally and a restrictive phenotype distally. This is not what was seen. Instead an intermediate phenotype in all regions of the wing is found. This result argues that fz functions in all regions of the wing at the same time and is not consistent with fz functioning in the propagation of a signal down the wing (Adler, 2000).

The complementary nature of the domineering nonautonomy of fz and Vang clones is striking. It is true for the anatomical direction of the non-autonomy (i.e. distal vs. proximal); the relationship of the domineering non-autonomy to the clone (i.e. affected wild-type hairs pointing toward or away form the clone), and for the interactions with pk mutations. It is suggested that the domineering non-autonomy of fz clones is a consequence of a failure of the clone cells to send a locally acting polarity signal. The domineering non-autonomy of Vang clones could be due to the Vang clone sending excess signal (models that reverse this arrangement are also possible) (Adler, 2000).

A model for tissue polarity signaling in the wing is presented. Early models to explain planar polarity in the insect epidermis suggested it could be a reflection of the vector of a concentration gradient and this idea has remained popular. It is suggested that a distal/proximal gradient of fz activity is produced in the early prepupal wing (or wing disc). One way this could be achieved is by a gradient of a Wnt (or other type of ligand) resulting in a gradient of ligand bound Fz. Later in development cells would produce a locally acting second signal in amounts proportional to Fz activity. This hypothetical signal is referred to as Z and it is suggested that ligand bound Fz activates more Z production than unbound Fz. In this way a gradient of Fz activity would be translated into a gradient of Z. Cells would respond by initiating prehair morphogenesis on the side of the cell where Z level was lowest. This would result in hair polarity being oriented in the same direction as the vector of the Z concentration gradient. This is consistent with previous results showing that a directed gradient of fz expression results in cells with higher Fz levels producing hairs that point toward cells of lower levels. The absence of fz activity in clone cells would result in no Z being produced by the clone and a local decrease in Z levels that would cause surrounding cells to produce hairs that point toward the clone as is observed. Such a model can effectively incorporate the affects of pk and ds mutations on the direction of fz domineering non-autonomy. Mutations in these genes could alter the relationship between the ligand bound state of fz and Z production. For example, in a new antimorphic dominant pk allele, pk^D wing unbound Fz receptor could act as a super-activator of Z production. This would lead to a reversed gradient of Z and to the reversal of both polarity and the direction of fz domineering non-autonomy. This model can also explain the observation that cells inside of a pk clone display the same polarity as do cells in a similar position in an entirely pk wing, since the alternative polarity caused by pk mutations would be due to abnormal amounts of Z. Such a model can also explain some of the results seen with Vang. The domineering non-autonomy of Vang could be due to Vang cells being constitutive for the production of high levels of Z. This would lead to locally elevated Z levels and cells surrounding Vang clones producing hairs that point away from the clone, as is observed. The model can also explain the ability of pk^D to enhance the extent of fz domineering non-autonomy and suppress the extent of Vang domineering non-autonomy. In the model the level of Z will be higher in all regions of a pk^D wing since now both bound and unbound Fz receptor will be strongly activating the production of Z. Thus, when a clone of cells lacking functional Fz protein is produced, the difference between the amount of Z produced by the clone cells and their neighbors will be increased over that seen in an otherwise wild-type wing. The ability of pk^D to inhibit the extent of domineering non-autonomy of Vang clones can be explained by the reduced difference in the level of Z produced by the clone and neighboring cells (Adler, 2000).

At first glance the model cannot explain the suppression of Vang domineering non-autonomy in a fz mutant background, because the clone should produce high levels of Z in a background where there is little or no Z produced. One possibility is that in the absence of functional Fz no Z can be produced. A second possibility is that fz has multiple functions in wing tissue polarity and that an additional function is what suppresses the domineering non-autonomy of Vang. The model can also explain the relatively weak and poorly penetrant domineering non-autonomy of pk clones. The cells in such clones would produce aberrant amounts of Z, however the difference between the normal and mutant levels would be less than is seen in a fz mutant clone (that produces no Z) or in a Vang mutant clone (that produces constitutive high levels of Z). Thus, it is reasonable that pk (and ds) clones would show weak domineering non-autonomy (Adler, 2000).

The results of the cell ablation experiments are also consistent with the model. After surgery wound healing took place in the pupal wing. To form a permanent hole it was necessary for the healing to juxtapose neighboring dorsal and ventral wing cells. In those cases the juxtaposed cells were of similar position along the proximal/distal axis and therefore would be expected to be producing similar levels of Z. Thus polarity disruptions, equivalent to a clone of fz cells that juxtapose cells that produce normal levels of Z with cells that produce none, would not be seen. The model also predicts that the domineering non-autonomy of fz clones should be greater in proximal regions and weaker in the most distal regions of the wing. Some evidence for this sort of variation was found, although over a large middle region of the wing no significant difference in the strength of domineering non-autonomy is seen. Perhaps the hypothesized gradient of Z is shallow in this region of the wing or the assay is not sensitive enough. The model also predicts that the domineering non-autonomy of Vang clones should be greater in distal and weaker in proximal regions, but this was not seen. The nature of the hypothesized factor Z and its receptor are unknown. The function of the genes that encode these factors should be required for cells to sense differences in fz activity. An attractive candidate for the receptor is fz itself, since previous experiments have indicated that fz has both cell non-autonomous and cell-autonomous functions in the development of wing tissue polarity. Two roles for fz are also suggested by experiments that found both Vang and starry night/flamingo are required for some, but not all fz functions. This could be explained by fz functioning both upstream and downstream of Vang and starry night. Alternative candidates for factor Z and its receptor are Delta and Notch. These genes have been shown to be downstream of fz in the eye and to interact with dishevelled (dsh) during wing development. An interesting feature of the model is that the fz-dependent production of factor Z should be dsh independent because dsh is acting cell autonomously. It is worth noting that models suggested to explain the development of ommatidial polarity also rely on two sets of gradients along the polar/equator axis. Indeed, there are substantial similarities between the models. One difference is that in contrast to this model for the wing, in the eye fz is suggested to be essential only in the read out of the secondary gradient (Adler, 2000).

In the dorsal thorax (notum) of the Drosophila pupa, the pI cell divides unequally with its spindle axis aligned with the anterior-posterior (a-p) axis of the fly body. It produces two different daughter cells, pIIa and pIIb. During this division, Numb and its adaptor protein Partner of Numb (Pon) form an anterior crescent and segregate unequally into the anterior pIIb cell. In fz mutant pupae, the division of the pI cell is oriented randomly relative to the a-p axis and the Numb crescent does form, but at a random position. Thus, Fz is not required to establish planar asymmetry per se, but is necessary to orient the axis of the asymmetric cell division. This indicates that additional genes may be required for establishing, rather than orienting, planar asymmetry in the pI cell (Bellaïche, 2001 and references therein).

Fz organizes the actin cytoskeleton at the site of hair formation. Planar polarity in the pI cell is established by a mechanism that involves a remodeling of the previously established apical-basal polarity. During the pI cell division, Baz and DaPKC relocalize from the apical cortex to the posterior lateral cortex, while Dlg and Pins accumulate asymmetrically at the anterior lateral cortex. This redistribution along the a-p axis leads to the formation of two complementary planar domains at the cell cortex. This mechanism of polarity establishment is distinct from the one described in Drosophila neuroblasts. In these cells, Pins is recruited via Insc by Baz to the apical cortex, and acts in a Dlg-independent manner to maintain the Baz/DmPAR-6/DaPKC/Insc complex at the apical cortex. Dlg interacts directly with Pins and regulates the localization of Pins and Baz. Pins acts with Fz to localize Baz posteriorly, but Baz is not required to localize Pins anteriorly. Finally, Baz and the Dlg/Pins complex are required for the asymmetric localization of Numb. Thus, the Dlg/Pins complex responds to Fz signaling to establish planar asymmetry in the pI cell (Bellaïche, 2001).

To test whether the initial Dlg-independent localization of Baz at the posterior cortex depends on Fz signaling, the distribution of Baz was studied in fz mutant pupae. In wild-type pupae, a clear accumulation of Baz is seen at the level of the AJ in 61% of the interphase pI cells. By contrast, an asymmetric distribution of Baz at the apical cortex is detected in only 19% of the interphase pI cells in fz mutant pupae. In the remaining 81% of the cells, the asymmetric accumulation of Baz is either weak or similar to that seen in the surrounding epithelial cells. This indicates that Fz signaling regulates the initiation of the asymmetric localization of Baz at the posterior cortex. At metaphase, however, Baz and Pins form misoriented crescents relative to the a-p axis that localize at opposite poles in fz mutant pI cells. It is concluded that the formation of the two opposite Baz and Pins domains does not depend on fz activity, and that planar asymmetry can be established in the absence of Fz signaling. However, as previously seen for pins, the asymmetric distribution of Baz is less pronounced in fz mutant pI cells than in wild-type cells. Moreover, Dlg is distributed around the entire cell cortex, indicating that Fz signaling is required for the anterior accumulation of Dlg (Bellaïche, 2001).

Since Pins localizes asymmetrically in a Fz-independent manner, it was asked whether Pins is necessary to localize Baz at one pole of the pI cell in the absence of Fz. It was found that Baz localizes uniformly around the basal-lateral cortex in 82% of the fz;pins double mutant pI cells at metaphase. Moreover, although Numb forms a crescent at anaphase in pI cells mutant for pins or fz, no Numb crescent is seen at either metaphase or anaphase in fz;pins double mutant pI cells. Consistently, loss of fz activity enhances the pins bristle loss phenotype. These data show that Pins and Fz act in a redundant manner to exclude Baz from the anterior cortex and to establish planar asymmetry in the pI cell (Bellaïche, 2001).

These results show that Pins localizes to the anterior cortex in a Baz-independent manner, in an orientation opposite that of Baz, as does Numb. Pins cooperates with Fz to exclude Baz from the anterior cortex of the pI cell. In contrast, in neuroblasts, Pins localizes in a Baz-dependent manner to the apical pole, opposite Numb, and stabilizes the Insc/Baz/DmPAR-6/DaPKC complex. Nevertheless, Pins promotes the localization of Numb in both cell types (Bellaïche, 2001).

One important difference between pI cells and neuroblasts is the lack of insc expression in pI cells. To test the functional significance of this lack of Insc, Insc was expressed in the pI cell. Under these circumstances, Insc and Pins localize at the anterior cortex. Insc triggers the anterior relocalization of Baz, while Numb forms a posterior crescent at anaphase. The pI cell division remains planar. This contrasts with the effect of Insc in epithelial cells. In these cells, Insc localizes apically and orients the spindle along the apical-basal axis. This further indicates that the apical-basal polarity is remodeled in the pI cell. It is concluded that the ectopic expression of Insc is sufficient to reverse the planar polarity axis of the pI cell and to modify the activity of Pins relative to Baz. In the absence of Insc, the Dlg/Pins complex excludes Baz, while expression of Insc leads to the formation of a Pins/Insc/Baz complex. In both cases, Numb localization is opposite that of Baz (Bellaïche, 2001).

During planar polarization of the Drosophila wing epithelium, the homophilic adhesion molecule Flamingo localizes to proximal/distal cell boundaries in response to Frizzled signaling; perturbing Frizzled signaling alters Flamingo distribution, many cell diameters distant, by a mechanism that is not well understood. diego, a tissue polarity gene, encodes a protein comprised of six ankyrin repeats that colocalizes with Flamingo at proximal/distal boundaries. Diego is specifically required for polarized accumulation of Flamingo and drives ectopic clustering of Flamingo when overexpressed. It is suggested that Frizzled acts through Diego to promote local clustering of Flamingo, and that the clustering of Diego and Flamingo in one cell nonautonomously propagates to others. Localized Frizzled signaling would modify the properties of the Diego protein at proximal-distal boundaries, increasing Diego's ability to promote clustering of itself and Flamingo. A bias in Diego localization might then be enhanced via homophilic interactions between Flamingo molecules on adjacent cells. This model is consistent with the reciprocal effects that Flamingo and Diego have on each other's localization (Feiguin, 2001).

Is Frizzled activity required for polarized Diego localization? Diego localization was examined in clones of frizzled null tissue. Diego protein is undetectable at the cortex between frizzled mutant cells. In contrast, Diego accumulates to a higher level at boundaries between wild-type and frizzled mutant cells, although it is not possible to resolve whether Diego is present on both sides of the boundary. These data suggest that Diego accumulates at boundaries between cells with different levels of Frizzled signaling activity (Feiguin, 2001).

Frizzled mutant clones nonautonomously reorganize the polarity of hairs distal to the mutant tissue, and the effect is most pronounced on the medial side of the clone. Correspondingly, Diego localization in cells on the distal/medial side of frizzled clones is reoriented, suggesting that perturbations in Frizzled signaling can nonautonomously repolarize the distribution of Diego (Feiguin, 2001).

To confirm that Diego localizes to boundaries between cells with different levels of Frizzled signaling activity, its distribution was examined in wings expressing Frizzled under the control of ptc:GAL4. This produces levels of Frizzled protein that change with distance from the AP boundary. In these wings, Diego relocalizes to reflect the artificial gradient of Frizzled expression generated by Ptc:GAL4. In the cells posterior to Frizzled-overexpressing cells, Diego is also relocalized to reflect the nonautonomous disruption of polarity caused by Frizzled. Taken together, these data show that apposition of cells with different levels of Frizzled causes the accumulation of Diego at the boundary between cells. These data further show that the nonautonomous disruption of polarity produced by altering Frizzled signaling results in the mispolarization of Diego (Feiguin, 2001).

Genetic evidence that Drosophila frizzled controls planar cell polarity and Armadillo signaling by a common mechanism

The frizzled (fz) gene in Drosophila controls two distinct signaling pathways: it directs the planar cell polarization (PCP) of epithelia and it regulates cell fate decisions through Armadillo (Arm) by acting as a receptor for the Wnt protein Wingless (Wg). With the exception of dishevelled (dsh), the genes functioning in these two pathways are distinct. A genetic approach, based on a series of new and existing fz alleles, has been taken for identifying individual amino acids required for PCP or Arm signaling. For each allele, attempts were made to quantify the strength of signaling by phenotypic measurements. For PCP signaling, the defect was measured by counting the number of cells secreting multiple hairs in the wing. Each allele was then examined for its ability to participate in Arm signaling by the rescue of fz mutant embryos with maternally provided fz function. For both PCP and Arm signaling a broad range of phenotypes was observed, but for every allele there is a strong correlation between its phenotypic strength in each pathway. Therefore, even though the PCP and Arm signaling pathways are genetically distinct, the set of signaling-defective fz alleles affected both pathways to a similar extent. This suggests that fz controls these two different signaling activities by a common mechanism. In addition, this screen yielded a set of missense mutations that identify amino acids specifically required for fz signaling function (Provelones, 2005a).

This study surveyed a representative set of new and existing fz mutant alleles and asked whether the two pathways to which fz can contribute, PCP and Arm signaling, can be separated genetically. Such an approach has been taken for other receptors, including the platelet-derived growth factor receptor, where a set of site-directed mutants discriminates between the various downstream effectors. This study took advantage of the relative ease by which new endogenous alleles of genes can be isolated in Drosophila. This allowed examination of the defects caused by mutant alleles without relying on gene transfer methods. Moreover, in vivo rather than cell culture gene function was tested; cell culture tests were the only approach by which Fz mutations have previously been examined for Wnt signaling function (Provelones, 2005a).

The main finding is that it was not possible to separate PCP and Arm signaling activity. While it cannot be excluded that pathway-specific alleles would be uncovered in a larger series, the data support the view that the mechanism by which Fz operates is similar in both pathways. This conclusion is based on the correlation in signaling activity, in particular the finding that several alleles behave as intermediate or weak in both assays. In general, such a correlation suggests that one activity of a gene is related to another activity in a mechanistic and functional way. The analysis was restricted to measurements in vivo. Therefore, the data cannot be quantified in such a way that would allow a statement that the two functions are linearly (or otherwise) related. However, the data suggest that the phenotypes display, at least by approximation, a semilinear relationship to each other. Therefore, it is proposed that Fz engages a component shared by both PCP and Arm signaling (Provelones, 2005a).

Elucidating the identity of this shared component is crucial to understanding how this receptor activates signaling. Defining amino acids in Fz specifically required for signaling could potentially provide a valuable means to probe Fz interactions. Previous attempts to generate signaling-compromised mutations in Fz have been restricted to examining only the intracellular amino acids. Because alleles of the endogenous gene were made, it was possiable to sample the entire Fz protein. Importantly, signaling-specific mutations were found in the extracellular loops and transmembrane helices in addition to the intracellular loops. Because these mutations are located throughout the entire protein, it is argued that Fz is a dynamic molecule that changes conformation to relay its activation to a shared intracellular signal component. There are two major candidates for this shared component: Dsh and Galpha_o. Both proteins have been implicated in PCP and in Arm signaling and the Dsh protein has been shown to bind Fz. How Dsh and Galpha_o are coupled to Fz is not yet clear (Provelones, 2005a).

The following model is proposed as the way in which the Fz protein controls two different signaling pathways. Fz signals in a ground state to the PCP pathway in the absence of a Wnt. Consistent with this model, it was found that none of the fz missense mutations, which were all isolated on the basis of their PCP phenotype, are located in the CRD. This model is also supported by recent work suggesting that Fz does not require a Wnt ligand in PCP signaling but that its activity is regulated by interactions between neighboring cells and differential levels of the cytoplasmic mediators Pk and Dsh. Furthermore, experiments with chimeric fz transgenes have shown that the transmembrane portion of Fz, not the CRD, is responsible for coupling it to PCP signaling. In this context, it should also be mentioned that global signaling during PCP may be under the control of differential expression of molecules involved in cell adhesion (Provelones, 2005a).

It is suggested that the presence of a Wnt ligand switches Fz to the Arm signaling pathway by recruiting to the pathway the membrane protein Arr. It has been previously shown that arr mutants disrupt Arm signaling but do not have a PCP phenotype. While the exact mechanism of Arr-Fz-Wg interactions is not clear, a currently favored hypothesis is that Arr acts together with Fz as a coreceptor for Wg, generating a ternary complex. On the cytoplasmic side, Arr recruits Axn to the membrane. It is speculated that the membrane localization of Axn via Arr brings it in close proximity with Dsh, resulting in the inactivation of Axn and the stabilization of Arm. However, it is also possible that recruiting Axn to the membrane via Arr is sufficient to drive signaling. It has also been proposed that Wg initiates signaling by recruiting Axn to the membrane via Dsh and that once at the membrane Axn is inactivated, allowing Arm to accumulate. Axn is a negative regulator of Arm signaling and, like arr, axn mutants have no polarity phenotype. During PCP signaling, in the absence of Wg, a trimeric complex between Wg, Fz, and Arr would not be assembled and Fz functions independently of Arr but also of Axn (Provelones, 2005a).

It is postulated that the presence of ligand-unbound Fz facilitates Dsh activation in PCP signaling. In contrast to arr and axn, dsh is required for both the Wg and the PCP pathway, making fz and dsh the only two genes shared between Wg and PCP signaling. The pathways are genetically distinct, however, in that there are three specific alleles of dsh that prevent it from participating in the PCP pathway but allow it to function normally in Arm signaling. These dsh alleles contain missense mutations that map to the same domain of the protein. This study has shown that, unlike dsh, the two functions of fz are not separable by specific mutations. This suggests that Fz activates a common component and that the switch between the two pathways occurs downstream of fz (Provelones, 2005a).

There is one other series of endogenous fz mutant alleles: mutations in the human Frizzled 4 (FZD4) gene led to familial exudative vitreoretinopathy (FEVR). The signaling pathway used by FZD4 is not entirely clear. Some studies suggest that FZD4 signals through an alternative (Ca²⁺) Wnt pathway. However, the finding that the mutations in the Wnt coreceptor LRP, which is implicated in Arm/ß-catenin signaling and not in the Ca²⁺ pathway, also cause FEVR does not support this model. Moreover, FZD4 in collaboration with LRP5 can be stimulated to activate Arm/ß-catenin signaling by Norrin, a non-Wnt ligand that binds to the CRD. Of the 12 FEVR alleles, four result in mutations in the CRD. Of the remaining alleles, one is located N terminal to the CRD (G36D), thus potentially interfering with the processing of the hydrophobic signal peptide. Another mutation (C181R) affects a conserved cysteine residue in the 'hinge' region. There is a mutation (R417Q) in a conserved arginine in the third intracellular loop and a stop codon (W319X) identical to the truncation allele fz^RN described in this study. Interestingly, there is a FEVR allele (G488D) that affects a conserved glycine residue in the seventh transmembrane domain that is also altered in the fz alleles fz^MP (G545E, this article) and fz^HC52 (G545R). Three FEVR alleles, S495F and Q505X and the in-frame deletion of MW493-4 , are located at the junction of the seventh transmembrane domain and the cytoplasmic tail near a conserved motif that has been shown to be required for Arm/ß-catenin signaling function. The existence of CRD mutations in FEVR alleles of FZD4 demonstrates that mutations in this domain can indeed have a functional consequence and highlights the role of the CRD in FZD4 function in the retina. It also supports the hypothesis that CRD mutations have not been recovered in any of the PCP screens for fz alleles because this domain is dispensable in that process (Provelones, 2005a).

The role of the cysteine-rich domain of Frizzled in Wingless-Armadillo signaling

The Frizzled (Fz) receptors contain seven transmembrane helices and an amino-terminal cysteine-rich domain (CRD) that is sufficient and necessary for binding of the Wnt ligands. Recent genetic experiments have suggested, however, that the CRD is dispensable for signaling. fz CRD mutant transgenes were generated and tested for Wg signaling activity. None of the mutants was functional in cell culture or could fully replace fz in vivo. Replacing the CRD with a structurally distinct Wnt-binding domain, the Wnt inhibitory factor, reconstitutes a functional Wg receptor. It is therefore hypothesized that the function of the CRD is to bring Wg in close proximity with the membrane portion of the receptor. This model was tested by substituting Wg itself for the CRD, a manipulation that results in a constitutively active receptor. It is proposed that Fz activates signaling in two steps: Fz uses its CRD to capture Wg, and once bound Wg interacts with the membrane portion of the receptor to initiate signaling (Povelones, 2005b).

The principle finding of this study is that the Fz CRD is required for efficient Arm signaling. Fz transgenes carrying CRD mutations have compromised Arm signaling function in cell culture and cannot fully restore Arm signaling to fz,fz2 mutants in vivo. In addition, adding a heterologous Wnt-binding domain (WIF) to a CRD-deleted fz restores its ability to activate Arm signaling via Wg in cell culture. Based on the manipulations and results, it is hypothesized that the function of the CRD is to bring Wg in close proximity with the membrane portion of the receptor, a function that can be taken over by other Wnt-binding domains. This idea was tested by creating a transgene fusing Wg to Fz, eliminating the CRD in the process; this results in a constitutively active receptor (Povelones, 2005b).

While both in vivo and in vitro tests reveal that mutants with a defective Wnt interaction domain are compromised for Arm signaling, the requirement for the CRD is most evident in cell culture where all of the mutants show a reduced activity, particularly the one where the entire CRD is lacking. In the cell culture experiments, where the Wg signaling can be measured in a quantitative manner, a range of responses were found to the CRD mutants, corresponding to the differences in Wnt-binding strength. A range of phenotypes was noticed after examining cuticles in vivo and the abilities of the CRD mutants to restore signaling. While these rescue data are more difficult to measure, the phenotypes correspond in strength to the in vitro signaling levels. It is inferred from this relationship that signaling operates through the same mechanism in vivo as in cell culture. As an extension of this argument, it is suggested that the CRD plays a similar role in cell culture as in the embryo. However, signaling in vivo is less stringently dependent on the presence of the CRD, suggesting that its absence is being compensated for by other factors. If the function of the CRD (or other Wnt-binding domains such as the WIF) is, as proposed, to bring Wg in close proximity to the membrane domain of Fz, it is possible this function is taken over by other molecules acting in trans and that these factors are not present in vitro. Candidates for such molecules are members of the CRD containing ROR family and the RYK receptor tyrosine kinase, which has a WIF domain. It is also possible that extracellular matrix molecules provide such an accessory function, by presenting or concentrating Wg close to the Fz signaling domain (Povelones, 2005b).

Is the only function of the CRD (or another Wg-binding domain, such as WIF) to capture Wg and to present it to the coreceptor Arrow? In that view, there would be no need for the seven-transmembrane domain of the Fz receptors; Fz would solely act to promote Wg interacting with Arrow. This was found to be unlikely; there are several studies that point to a requirement of specific residues in the Fz membrane domain in signaling. Mutations in those residues, either engineered or present in natural alleles, disrupt signaling. In addition, it has been recently proposed that in Drosophila, fz activates PCP and Arm signaling through heterotrimeric G proteins. Finally, expressing the CRD on the cell's surface as a GPI-linked membrane molecule does not promote signaling, but instead acts as a dominant negative. Taken together, these data suggest that the transmembrane portion of fz is a dynamic signal activating molecule and not merely a Wg presentation module (Povelones, 2005b).

Overexpression of fz^WIF in the Drosophila wing leads to both gain-of-function PCP and Arm signaling phenotypes. This is the composite of the consequences of fz and fz2 overexpression, which individually activate PCP and Arm signaling, respectively. There is much interest in determining how each receptor couples to a particular pathway. Although there is some disagreement in these studies, it is generally concluded that the transmembrane portion of fz, including the cytoplasmic tail, couples it to PCP signaling. Since fz^WIF contains this portion of fz, it is not surprising that it too affects PCP signaling. What structural feature of fz2 is responsible for coupling it exclusively to Arm signaling? It was found that specifically replacing the fz CRD with the WIF domain results in a receptor that, like fz2, can activate Arm signaling. This finding is consistent with a study of fz/fz2 chimeras where the ability to activate Arm signaling was shown to be a property of the fz2 CRD. It was proposed that the feature conferring Arm coupling was the 10-fold higher affinity of the fz2 CRD for the Wg protein. By analogy, the WIF domain, like the fz2 CRD, may have a higher affinity for Wg than the fz CRD (Povelones, 2005b).

The roles of glypicans Dally and Dally-like protein (Dlp), the Wg receptors Frizzled (Fz) and Fz2, and the Wg co-receptor Arrow (Arr) in Wg gradient formation in the wing disc

During the wing development Wingless acts as a morphogen whose concentration gradient provides positional cues for wing patterning. The molecular mechanism(s) of Wg gradient formation is not fully understood. This study systematically analyzes the roles of glypicans Dally and Dally-like protein (Dlp), the Wg receptors Frizzled (Fz) and Fz2, and the Wg co-receptor Arrow (Arr) in Wg gradient formation in the wing disc. Both Dally and Dlp are essential and have different roles in Wg gradient formation. The specificities of Dally and Dlp in Wg gradient formation are at least partially achieved by their distinct expression patterns. Surprisingly, although Fz2 has been suggested to play an essential role in Wg gradient formation by ectopic expression studies, removal of Fz2 activity does not alter the extracellular Wg gradient. Interestingly, removal of both Fz and Fz2, or Arr causes enhanced extracellular Wg levels, which mainly results from upregulated Dlp levels. It is further shown that Notum, a negative regulator of Wg signaling, downregulates Wg signaling mainly by modifying Dally. Last, it is demonstrated that Wg movement is impeded by cells mutant for both dally and dlp. Together, these new findings suggest that the Wg morphogen gradient in the wing disc is mainly controlled by combined actions of Dally and Dlp. It is proposed that Wg establishes its concentration gradient by a restricted diffusion mechanism involving Dally and Dlp in the wing disc (Han, 2005).

One important finding of this study is that removal of the Wg receptors (Fz and Fz2) and the co-receptor Arr does not lead to a loss of extracellular Wg. Fz2 has been proposed to play a major role in Wg gradient formation in the wing disc by ectopic expression studies. Although the high capacity of Fz2 in stabilizing Wg has been demonstrated, loss-of-function results clearly show that extracellular Wg levels are not reduced in clones mutant for fz2. This is apparently not due to the overlapping function of Fz, since the extracellular Wg level is enhanced, rather than reduced, in the absence of both Fz and Fz2 functions. The results argue that Fz2 is not essential for extracellular Wg gradient formation in vivo. It is important to note that in addition to Fz and Fz2, Fz3 is also expressed in the wing disc and its expression is upregulated by Wg signaling. Although Fz3 has lower affinity than Fz2 in Wg binding and acts as an attenuator of Wg signaling, its role in Wg distribution needs to be determined (Han, 2005).

It is further demonstrated that extracellular Wg is enhanced in cells mutant for fz-fz2 or arr, suggesting that Wg receptors (Fz and Fz2) and Arr shape extracellular Wg gradient by downregulating extracellular Wg levels. The data argue that this mainly results from upregulation of Dlp. Consistent with this view, the accumulated extracellular Wg can be eliminated by loss of HSPGs in sfl-fz-fz2 or arr-botv mutant clones. Importantly, it is shown that both extracellular Wg and Dlp levels are upregulated on the cell surface of clones mutant for dsh. These data provide compelling evidence that though a feedback mechanism, Wg signaling can control the Dlp levels to regulate the extracellular Wg gradient (Han, 2005).

Another alternative possibility is that enhanced Wg levels in fz-fz2 or arr clones may be caused by impaired Wg internalization. Although a significant amount of internalized Wg vesicles has been demonstrated in fz-fz2 or arr mutant clones, this possibility cannot be ruled out, since a quantitative comparison of Wg internalization between wild-type cells and fz-fz2 or arr mutant cells is difficult. Furthermore, as mentioned above, Fz3 is expressed in the wing disc and its expression is upregulated by Wg signaling. It is possible that Fz3 may mediate the internalization of Wg in the absence of Fz and Fz2 (Han, 2005).

Evidence has been presented that Wg morphogen movement is regulated by a diffusion mechanism(s) in the wing disc. Does Wg diffuse freely in the extracellular matrix/space? In this work, it is shown that Wg fails to move across a strip of cells mutant for the HSPGs Dally and Dlp. This result suggests that Wg cannot freely diffuse in the extracellular matrix. Instead, the findings are consistent with a model in which Wg movement is mediated by the HSPGs Dally and Dlp through a restricted diffusion along the cell surface. Similar mechanisms have been proposed for Hh and Dpp. In biological systems such as imaginal discs, the restraint of Wg spreading to the surface of the epithelial cell layer is important since the folding of imaginal discs, such as the leg disc, poses a problem if the Wg gradient formation were to occur out of the plane of the epithelial cell layer through free diffusion. In agreement with this view, the model proposes that Wg gradient formation depends on Wg movement through the cell surface of the disc epithelium (Han, 2005).

Hexagonal packing of Drosophila wing epithelial cells by the planar cell polarity pathway

The mechanisms that order cellular packing geometry are critical for the functioning of many tissues, but they are poorly understood. This problem was investigated in the developing wing of Drosophila. The surface of the wing is decorated by hexagonally packed hairs that are uniformly oriented by the planar cell polarity pathway. They are constructed by a hexagonal array of wing epithelial cells. Wing epithelial cells are irregularly arranged throughout most of development, but they become hexagonally packed shortly before hair formation. During the process, individual cell boundaries grow and shrink, resulting in local neighbor exchanges, and Cadherin is actively endocytosed and recycled through Rab11 endosomes. Hexagonal packing depends on the activity of the planar cell polarity proteins. It is proposed that these proteins polarize trafficking of Cadherin-containing exocyst vesicles during junction remodeling. This may be a common mechanism for the action of planar cell polarity proteins in diverse systems (Classen, 2005).

A link between the PCP pathway and epithelial repacking is suspected, because repacking occurs at the time that these proteins are thought to polarize. Therefore neighbor number and junction length variability was quantified at the time of hair outgrowth in different PCP mutants. For prickle (pk-sple^13/26), neighbor number was quantitated over time (Classen, 2005).

pk-sple^13/26 wings begin repacking at the same time as wild-type; however, the process is less successful. Whereas wild-type wings reduce the percentage of pentagonal cells from 34% to 13% by the time that hairs begin to emerge, pk-sple^13/26 wings retain 21%. Thus, about 40% of the pentagonal cells that normally assemble boundaries with new neighbors (and become hexagonal) fail to do so in pk-sple mutants. Consistent with this, pk-sple wing epithelia contain abnormally high numbers of four-way vertices between cells. pk¹ mutant wings are even more irregularly packed than pk-sple^13/26 wings. A total of 62% of the pentagonal cells that would normally become hexagonal fail to assemble boundaries with new neighbors in pk¹ wings. Even four-sided cells accumulate significantly in pk¹ mutant wings. Individual cell contact lengths are also much more variable; while pk-sple^13/26 boundary lengths were 9% more variable than wild-type, those of pk¹ were 42% more variable. These data are consistent with the earlier observation that adult pk wings frequently contain pentagonal cells. These data suggest that the assembly of new cell boundaries and regularization of junction length do not occur efficiently in the absence of products of the Pk-Sple locus (Classen, 2005).

Packing defects of the hypomorphic Flamingo (fmi) allele, fmi(stan)³, are mild but significant. The null allele fmi^E59 produces much stronger defects. The variability of individual junctional lengths in these cells is more than twice that of wild-type, and only 69% of fmi^E59 mutant cells become hexagonal, compared with 78% in wild-type. Pentagonal cells persisted in fmi^E59 mutants (27% compared with 13% in wild-type). This suggests that the majority of pentagonal cells fail to assemble boundaries with new neighbors when Fmi is missing (Classen, 2005).

The packing geometry was examined of two different frizzled (fz) alleles, fz^R52 and fz^P21. fz^P21 mutant wings fall into two classes. While the majority of wild-type and PCP mutant wings initiate hair formation by 42 hr after puparium formation (APF) (at 22°C), a subset of fz^P21 mutant wings does not. Since these wings were not apoptotic (as indicated by Caspase staining), they were included in the analysis and quantified separately. Even at 50 hr APF, their packing is much more irregular than that of wild-type . Defects in fz^P21 mutant wings that do initiate hair formation by 42 hr APF are milder but still significant. fz^R52 homozygotes do not produce viable pupae in these experiments, and homozygous mutant clones are small. These clones have even stronger packing defects than those of fz^P21, suggesting that little repacking occurs in fz^R52 homozygous tissue. Thus, Fz is needed to develop regular hexagonal packing (Classen, 2005).

stbm⁶ and dgo³⁸⁰ mutant wings have milder, but significant, alterations in the ratio of pentagons, hexagons, and heptagons and of four-way vertices. Both mutants, however, affect junction length variability more strongly than pk-sple^13/26. Taken together, these data indicate that PCP mutant cells fail to efficiently assemble boundaries with new neighbors and cannot regularize their packing geometry (Classen, 2005).

To ask whether interfering with PCP polarity could alter the geometry of packing in wild-type cells, cells were examined surrounding PCP mutant clones with either autonomous (fmi^E59) or nonautonomous (fz^R52) effects on polarity. The frequency of pentagons, hexagons, and heptagons was examined in fz^R52 and fmi^E59 mutant clones, and in the areas of disturbed and normal Fmi polarity surrounding both. The mutant cells within both fz^R52 and fmi^E59 clones are abnormally packed. However, whereas the packing defects caused by Fmi clones are predominantly restricted to the clone and directly adjacent cells, Fz clones alter packing over long distances in wild-type tissue in the same regions where Fmi polarity is disturbed. The abnormal packing of wild-type cells surrounding fz^R52 clones is unlikely to be a consequence of altered cell packing within the mutant clone, because fmi^E59 mutant clones pack just as abnormally, but do not perturb packing in the surrounding tissue. This suggests that dominant reorientation of Fmi polarity by frizzled mutant clones disturbs the repacking of wild-type cells (Classen, 2005).

To investigate how the PCP proteins were localized during repacking, pupal wings were imaged for Fmi before, during, and after hexagonal packing. Since it is thought that PCP proteins do not polarize until shortly before hair formation, it was surprised to find that the subcellular distribution of Fmi is polarized in many areas of the wing before junction remodeling is initiated, even in late third instar wing discs and prepupal wings. Fz-GFP is distributed similarly. This polarity may have been missed because it exhibits less long-range coherence in imaginal discs and prepupal wings than it does later (Classen, 2005).

In prepupal wings, Fmi polarity is roughly proximal-distal in the region surrounding L3. Coherent Fmi polarity is lost at the beginning of the pupal period: this is exactly the time at which junction remodeling initiates. Although polarity is not coherent, Fmi is not uniformly distributed along cell boundaries. This can be clearly seen when Fmi localization is compared to that of E-Cadherin (Classen, 2005).

At pupal time TP1, Fmi polarization begins in vein cells as they contract their apical cross-section. Intervein regions contain only small groups of cells with coherent polarity, and the axes of these groups are not always proximal-distal. By TP2, Fmi polarity is coherent between larger groups of cells, although the axis of polarity is still mixed. Fmi polarity is aligned in large coherent domains along the proximal-distal axis by TP4, when hexagonal packing is completed, and it remains unchanged at TP5 when hairs emerge. In summary, PCP proteins polarize during larval and prepupal stages, alignment of polarity between cells is disturbed when junction remodeling begins, and long-range polarity is reestablished as hexagonal packing is completed. Early polarization of PCP proteins is consistent with the genetic requirement for fz and ds activity at this time to determine the axis of polarity, and it suggests that the feedback loop that organizes coupled proximal and distal domains probably acts during these early stages (Classen, 2005).

It was asked whether PCP proteins might affect packing by influencing recycling of junctional components. Therefore, it was asked whether PCP mutants enhance the hole formation caused by shi loss of function. Double mutant pupae were shifted to a subrestrictive temperature that never causes holes to form in shi mutants or in PCP mutants. When shi is combined with dgo³⁸⁰, stbm⁶, stbm¹⁵³, stbm^D, stan³, pk-spl¹, or pk¹, hole formation occurs even under these mild conditions. This raises the possibility that PCP proteins may worsen Cadherin recycling defects in dynamin mutant cells. Consistent with this, gaps in Cadherin arise more frequently in double shi;pk¹ or shi;dgo³⁸⁰ mutant wings than in wings mutant for shi alone. This suggests that Cadherin is recycled less efficiently in the absence of PCP proteins (Classen, 2005).

Despite this enhancement, no striking abnormalities in Cadherin distribution were seen in most PCP mutants. fz^P21 mutant cells sometimes show gaps in E-Cadherin that are similar to, but much less frequent than, those of shi mutants. In fmi^E59 mutant cells, E-Cadherin levels are elevated, but no gaps in localization are observed. These observations suggest that PCP proteins are not required for delivery of Cadherin to cell contacts during remodeling. Nevertheless, the PCP mutants enhance Cadherin recycling defects caused by loss of Dynamin. One model consistent with this shows that PCP proteins bias Cadherin recycling to specific places on the cortex. Reducing both the rate of recycling and its elevation at a particular site could exacerbate the failure of Cadherin delivery to growing cell boundaries (Classen, 2005).

To test whether exocyst components were polarized by PCP proteins, Sec5 localization was examined during repacking of the wing epithelium. At this time, cell shapes are irregular, and Fmi polarity is not coherent between cells. Nevertheless, Fmi accumulates preferentially on specific regions of the cortex. Although Sec5 vesicles are seen throughout the cell, they are particularly enriched near Fmi-positive cell boundaries. Enrichment persists as Fmi polarity becomes aligned (Classen, 2005).

To test whether Fmi plays an active role in recruiting Sec5, Fmi was overexpressed and Sec5 localization was examined. Overexpressed Fmi is present uniformly around the cortex and in large punctate structures within the cell. Sec5 dramatically accumulates in cells overexpressing Fmi and is recruited to sites of Fmi localization. Large internal structures positive for Fmi and Sec5 also contain Cadherin. These observations indicate that Fmi can recruit Sec5-positive vesicles containing E-Cadherin, and they suggest that PCP proteins may promote hexagonal packing by polarizing membrane trafficking (Classen, 2005).

The conserved cassette of PCP proteins controls a variety of seemingly different developmental processes, and no common cell biological mechanism has ever been proposed for their action. Polarizing membrane trafficking by recruiting Sec5 is a basic function that could be utilized in many different contexts, and it may help explain the requirement of PCP proteins in a divergent set of processes. Both rotation of photoreceptor clusters and convergent extension movements depend on the ability of cells to make and break intercellular contacts, as they do during hexagonal packing in the wing. Consistent with this, Silberblick (Wnt-11) acts through the PCP pathway and appears to affect endocytic trafficking of Cadherin during zebrafish gastrulation. Recruitment of exocyst components might also be a plausible mechanism to explain the ability of PCP proteins to bias Notch Delta signaling between R3 and R4 photoreceptors, since Delta delivery is dependent on the exocyst. In the future, identifying the chain of events that leads from PCP protein localization to exocyst recruitment may increase the understanding of these important processes (Classen, 2005).

Planar polarity genes in the Drosophila wing regulate the localisation of the FH3 domain protein Multiple Wing Hairs to control the site of hair production

The core planar polarity proteins play important roles in coordinating cell polarity, in part by adopting asymmetric subcellular localisations that are likely to serve as cues for cell polarisation by as yet uncharacterised pathways. This study describes the role of Multiple Wing Hairs (Mwh), a novel Formin Homology 3 domain protein, which acts downstream of the core polarity proteins to restrict the production of actin-rich prehairs to distal cell edges in the Drosophila pupal wing. Mwh appears to function as a repressor of actin filament formation, and in its absence ectopic actin bundles are seen across the entire apical surface of cells. The proximally localised core polarity protein Strabismus acts via the downstream effector proteins Inturned, Fuzzy and Fritz to stabilise Mwh in apico-proximal cellular regions. In addition the distally localised core polarity protein Frizzled positively promotes prehair initiation, suggesting that both proximal and distal cellular cues act together to ensure accurate prehair placement (Strutt, 2008).

Activity of the core planar polarity proteins is required in cells of the Drosophila pupal wing to specify prehair initiation at the distal vertex (Wong, 1993). This study presents evidence that core polarity protein localisation at both proximal and distal cell edges provides redundant cues for specifying distal prehair initiation (Strutt, 2008).

Regarding the mechanistic basis of the proximal cue, this and previous work provide evidence for a plausible model. The downstream effectors In, Fy and Frtz all colocalise at the proximal cell edge with Stbm and in a Stbm-dependent manner. Activity of In, Fy and Frtz is required for Mwh phosphorylation and its subapical subcellular localisation, which is thus concentrated towards the proximal side of the cell. Genetic studies have shown that loss of fy, in, frtz or mwh activity leads to excess prehair initiation, and this study found that the initial defect in mwh is excess actin bundling across the entire apical face of cells. Thus, proximal restriction of Mwh activity in the cell results in actin bundling and prehair initiation specifically in distal regions (Strutt, 2008).

Additional evidence for the sufficiency of a Stbm-dependent cue for prehair initiation at opposite cell edges comes from experiments in the abdomen. It was reported that cells lacking fz activity, but juxtaposed to cells with fz activity, could produce polarised trichomes, as has also been observed in the first row of cells within a fz clone in the wing (Strutt, 2008).

Less information is available regarding the distal cue. Its existence is based upon two pieces of evidence. First, if prehair initiation were entirely dependent on Stbm-mediated localisation of Mwh activity, then prehairs should show no bias in their site of initiation in cells lacking stbm activity. In fact, stbm mutant cells with Fz localised at one cell edge show a strong bias towards initiating prehairs at this edge. Second, if prehair initiation is controlled only by a Stbm-dependent repressive cue, then in the absence of stbm activity, Fz would have no influence over prehair initiation. Instead, in a stbm background, fz activity still weakly promotes prehair formation. Taken together these data support the view that distally localised Fz acts as a prehair promoting cue (Strutt, 2008).

A possible mechanism of action of the distal cue would be if localised Fz were able to repress Mwh activity in distal cell regions, possibly via its known effectors RhoA and Drok. Alternatively Fz could promote prehair initiation in a Mwh-independent fashion, either via RhoA/Drok or other effectors (Strutt, 2008).

It is notable that absence of fz activity results in a delay in prehair formation, and a greater tendency for prehairs to form in the cell centre rather than towards a cell edge, than loss of stbm. It is surmised that in fz mutant cells, there is no Fz-dependent prehair promoting cue, and the Stbm-dependent repressive cue is evenly distributed around the cell edge, resulting in delayed prehair initiation in the cell centre. Conversely, in stbm mutant cells, there is no change in the activity of the repressive cue, but the Fz-dependent prehair promoting cue is localised to cell edges, albeit more thinly spread than in the wildtype situation. This results in approximately normally timed prehair initiation near cell edges (Strutt, 2008).

An unexplained observation is that within stbm mutant tissue, the site of prehair initiation appears to be biased towards that seen in neighbouring cells. Thus in the first rows of cells within a clone, prehairs tend to point towards the adjacent wildtype tissue. This phenomenon is presumably independent of core protein asymmetric localisation, and may depend upon some mechanical linkage between cells. In this context, there is already evidence that the microtubule cytoskeletons of adjacent cells may be linked and that this could coordinate cell polarity (Turner, 1998). An alternative core protein-independent mechanism to align wing hairs, relying on the activities of Gliotactin and Coracle has also been reported (Strutt, 2008).

Loss of in, fy, and frtz results in a similar phenotype to loss of mwh with multiple ectopic prehairs at the cell edge preceded by excess apical actin bundling. As In, Fy and Frtz are all required for the apical punctate distribution of Mwh within cells, and also appear to stabilise each other, this suggests that In, Fy and Frtz act together to activate Mwh and promote apical localisation. Conversely, while Stbm plays a role in localising Mwh within the cell, it is not required for its activity, as loss of stbm does not phenocopy mwh mutants in which increased apical actin bundling is observed. This role of Stbm in localising but not regulating Mwh activity is most simply explained by Stbm acting to localise, but not regulate In, Fy and Frtz activities. This is supported by the observation that whereas loss of fz or stbm has a strong effect on the distribution of Frtz to the apicolateral junctions, it has a negligible effect on the apparent phosphorylation state of Mwh (Strutt, 2008).

The regulation of Mwh activity appears to be largely post-translational; although the subcellular distribution of Mwh changes dramatically in frtz mutant cells, total levels of Mwh are not similarly altered. Further evidence that In, Fy and Frtz regulate Mwh activity by a mechanism largely independent of Mwh protein levels comes from the observation that Mwh overexpression in the wing produces no effect on trichome formation, rather than repressing trichome formation as might be predicted if protein levels were the main determinant of activity (Strutt, 2008).

The data are strongly suggestive that Mwh activity may be regulated by phosphorylation. Treatment of cell extracts with phosphatase results in increased mobility of Mwh. A similar increase in mobility is observed when frtz activity is removed, but not when stbm or fz activities are removed. Thus, at the least, Mwh phosphorylation correlates with Mwh activity and apical punctate localisation. Hence it is proposed that the rôles of In, Fy and Frtz may be to activate, or bring into proximity with Mwh, a kinase or kinases responsible for activating Mwh. Similarly, Fz could locally promote the dephosphorylation of Mwh to induce prehair initiation, although any such effect would have to be small, as Mwh phosphorylation is not obviously altered in the absence of Fz (Strutt, 2008).

Definitive proof that phosphorylation of Mwh is important for its activity would require the identification of particular phosphorylation sites which were required for specific molecular functions and/or identification of a kinase critically required for Mwh activity (Strutt, 2008).

An alternative regulatory mechanism for Mwh, via analogy to Diaphanous family formins, would be via RhoA GTPase activity. The FH2 domain of such formins promotes actin nucleation, an activity which is autoinhibited by interaction with the GTPase binding domain (GBD). Upon interaction of the GBD with GTPase-bound Rho GTPases, this autoinhibition is released. Notably, genetic interaction data suggest that Fz/Dsh can activate RhoA activity. This is consistent with a model whereby in the proximal cell Rho GTPase activity is low and Mwh inhibits prehair initiation, and in the distal cell activated RhoA alleviates the inhibitory activity of Mwh (Strutt, 2008).

Notwithstanding the evidence for post-translational regulation of Mwh activity in the normal context of the pupal wing, in cultured cells no effect is seen of Mwh overexpression on the actin cytoskeleton. This seems likely to be due to the much higher levels of expression that can be achieved in transfected cells as opposed to cells in the living organism, and hence the result should be treated with caution, but may suggest that S2 cells express a factor able to constitutively activate Mwh (Strutt, 2008).

The results also indicate that Mwh levels are influenced by temperature, which provides a plausible explanation for why in, fy and frtz phenotypes are stronger at 18°C. It is suggested that loss of in, fy and frtz reduces Mwh activity, and lower temperatures additively reduce Mwh levels, resulting in lower overall Mwh activity (Strutt, 2008).

What is the molecular function of Mwh? As already noted, the FH3 domain of conventional formins is thought to be involved in targeting the protein to particular cellular sites, whereas the GBD domain is involved in inhibition of the actin nucleating function of the FH2 domain. A plausible model is that Mwh acts as a dominant negative by binding via its GBD domain to other FH2 domain containing formins that are involved in the nucleation of actin filaments and inhibiting their activity. Notably, this dominant negative activity of Mwh could then be inhibited distally in the cell by Fz-mediated activation of RhoA GTPase activity (Strutt, 2008).

Electron microscopy studies suggest that prior to prehair initiation the apical cell surface is covered in electron-dense 'pimples' that are normally only activated at the distal cell edge and serve as foci for actin filament formation (Guild, 2005). It is proposed that at around 32 hours of pupal development, cells receive a general signal for pimple activation which results in actin nucleation, and that Mwh activity is required to inhibit this activation away from the distal cell edge (Strutt, 2008).

Frizzled function in retinal polarity

Effects of mutation - Continued: part 2/2

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