dachsous

REGULATION

Protein interactions

Four-jointed is a Golgi kinase that phosphorylates a subset of cadherin domains

The atypical cadherin Fat acts as a receptor for a signaling pathway that regulates growth, gene expression, and planar cell polarity. Genetic studies in Drosophila identified the four-jointed gene as a regulator of Fat signaling. This study shows that four-jointed encodes a protein kinase that phosphorylates serine or threonine residues within extracellular cadherin domains of Fat and its transmembrane ligand, Dachsous. Four-jointed functions in the Golgi and is the first molecularly defined kinase that phosphorylates protein domains destined to be extracellular. An acidic sequence motif (Asp-Asn-Glu) within Four-jointed is essential for its kinase activity in vitro and for its biological activity in vivo. These results indicate that Four-jointed regulates Fat signaling by phosphorylating cadherin domains of Fat and Dachsous as they transit through the Golgi (Ishikawa, 2008).

The Fat and Hippo signaling pathways intersect at multiple points and influence growth and gene expression through regulation of the transcriptional coactivator Yorkie. Fat signaling also influences planar cell polarity (PCP). Fat acts as a transmembrane receptor, and is a large (5147 amino acids) atypical cadherin protein, with 34 extracellular cadherin domains. Dachsous (Ds) is also a large (3503 amino acids) transmembrane protein with multiple cadherin domains and is a candidate Fat ligand because it appears to bind Fat in a cultured cell assay, acts non-cell autonomously to influence Fat pathway gene expression, and acts genetically upstream of fat in the regulation of PCP. A second protein, Four-jointed (Fj), also acts non-cell autonomously to influence Fat pathway gene expression and acts genetically upstream of fat in the regulation of PCP. However, Fj is a type II transmembrane protein that functions in the Golgi. Thus, Fj might influence Fat signaling by posttranslationally modifying a component of the Fat pathway (Ishikawa, 2008).

To investigate the possibility of modification of Fat or Ds, FLAG epitope-tagged fragments of their extracellular domains together were coexpressed with Fj in cultured Drosophila S2 cells. When the first 10 cadherin domains of Ds (Ds1-10) were coexpressed with Fj, a shift in mobility was observed. A common posttranslational modification of secreted and transmembrane proteins as they pass through the Golgi is glycosylation. Most glycosyltransferases contain a conserved sequence motif, Asp-X-Asp (DXD; X, any amino acid), which is essential for their activity. Because a related sequence motif [Asp-Asn-Glu (DNE) at amino acids 490 to 492] is present in Fj and its vertebrate homologs, a mutant form of Fj was created in which DNE was changed to GGG (Fj^GGG; G, glycine). The expression levels and Golgi localization of Fj^GGG appear normal, but Fj^GGG expression did not shift Ds1-10 mobility (Ishikawa, 2008).

To identify modified cadherin domains, smaller fragments of Ds1-10 were expressed. The smallest fragments whose mobility was shifted in cells expressing Fj were two-cadherin-domain polypeptides: Ds2-3, Ds5-6, and Ds8-9. Ds2-3 and Ds5-6 appeared to be stoichiometrically modified in cells expressing Fj, whereas Ds8-9 was only partially modified. Fat4-5 was also partially shifted by Fj coexpression. The mobility shifts of these two-cadherin-domain polypeptides were not observed with Fj^GGG. To identify potential sites of modification, their sequences were aligned. This identified four sites at which a Ser or Thr residue was conserved, whose hydroxyl groups could potentially be sites of posttranslational modification. To evaluate their influence, each was mutated in turn to Ala within the Ds2-3 polypeptide. Three of the four mutants had no effect; however, one, Ds2-3^S236A (mutation of Ser²³⁶ to Ala), completely eliminated the Fj-dependent mobility shift. Introduction of an analogous mutation into Ds8-9 also eliminated its mobility shift. Thus, a Ser reside at a specific location within the second of the two cadherin domains was essential for the Fj-dependent mobility shift. This amino acid was a Ser in each of these dicadherin domains, but Thr was also compatible with the Fj-dependent modification. In a structurally solved cadherin domain, this Ser is the seventh amino acid and predicted to be located on the surface near the middle of the cadherin domain (fig. S2) (Ishikawa, 2008).

To identify posttranslational modifications associated with this mobility shift, Ds2-3 was purified from S2 cells expressing or not expressing Fj, the proteins were digested with trypsin, andthe resulting peptides were analyzed by mass spectrometry. One peptide from Fj-expressing cells was stoichiometrically shifted by 80 daltons relative to the same peptide from cells not expressing Fj, and it also eluted earlier on high-performance liquid chromatography (HPLC). Mass and tandem mass spectrometry (MS/MS) fragmentation patterns identified this peptide as amino acids 215 to 237 of Ds and refined the site of modification to within amino acids 232 to 237. The mass of the equivalent peptide from Ds2-3^S236A was not altered by Fj expression. Most of the peptides corresponding to Ds2-3 cadherin domains were identified, and none of the others were detectably modified in cells expressing Fj. Thus, the Fj-dependent modification of Ds2-3 comprises an addition of 80 daltons, which is attached to Ser²³⁶. An 80-dalton mass does not correspond to that of any known glycans, but does correspond to the mass associated with addition of a phosphate group. Incubation of Fj-modified Ds fragments with either calf intestinal alkaline phosphatase (CIP) or Antarctic phosphatase (AnP) reversed the Fj-dependent mobility shifts of Ds2-3, Ds8-9, and Fat 4-5. Thus, Ds and Fat cadherin domains are subject to Fj-dependent phosphorylation at a specific Ser residue (Ishikawa, 2008).

To investigate whether Fj itself has kinase activity, a secreted, epitope-tagged Fj (sFj:V5) was purified from the medium of cultured S2 cells. Purified sFj:V5 was then incubated with affinity-purified Ds2-3 and [γATP (adenosine 5'-triphosphate)] in buffer. Transfer of ³²P onto Ds2-3 was observed in the presence of sFj, but not in its absence, and not when sFj^GGG was used as the enzyme. Moreover, Ds2-3^S236A was not detectably phosphorylated by sFj. The activity of Fj expressed in a heterologous system was also characterized by expressing a glutathione S-transferase:Fj (GST:Fj) fusion protein in Escherichi coli and partially purifying it on glutathione beads. GST:Fj, but not GST:Fj^GGG, catalyzed the transfer of ³²P onto Ds2-3. Thus, Fj is a protein kinase (Ishikawa, 2008).

The generic kinase substrates myelin basic protein and casein were not detectably phosphorylated by sFj. Thus, Fj appears to have a limited substrate specificity. Only a few proteins have been identified as being phosphorylated in the secretory pathway, and none of the responsible kinase(s) have been molecularly identified. A Golgi kinase activity, referred to as Golgi casein kinase, preferentially phosphorylates Ser or Thr residues within a S/T-X-E/D/S(Phos) consensus sequence. Because Fj does not phosphorylate casein, and the Ser residues within cadherin domains targeted by Fj do not conform to Golgi casein kinase sites, Fj is not Golgi casein kinase. Fj autophosphorylation was detected, but this reaction was weak compared to phosphorylation of Ds2-3. The autophosphorylation reaction is apparently unimolecular, because GST:Fj and sFj:V5 did not phosphorylate each other and the fraction of Fj phosphorylated was independent of concentration (fig. S5) (Ishikawa, 2008).

Some cadherin domain polypeptides that include a Ser as the seventh amino acid were not detectably shifted, but the mobility shift on Ds2-3 might reflect a conformational effect. To examine the ability of Fj to phosphorylate other cadherin domains, in vitro kinase reactions were performed with [γ-³²P]ATP. This identified phosphorylation sites on polypeptides that were not gel shifted, including Fat2-3, Fat10-11, and Fat12-13. The in vitro kinase reactions also identified differences in the efficiency with which different cadherin domains were phosphorylated by Fj, with Ft3, Ds3, and Ds6 being the best substrates (Ishikawa, 2008).

If the presence of a Ser or Thr at the seventh amino acid of a cadherin domain is taken as the minimal requirement for Fj-mediated phosphorylation, there are nine potential sites in Ds and 11 in Fat. However, Fat10, Ds2, Ds11, Ds13, and Ds18 were not detectably phosphorylated, despite the presence of Ser or Thr at this position. Presumably, there are other structural features important for recognition by Fj. This was also emphasized by the detection of phosphorylation of our Ds2-3 polypeptide, but not the Ds3-4 polypeptide, even though both contain Ser²³⁶. The dicadherin constructs were based on published annotations, but in comparing Ds cadherin domains to structurally solved cadherin domains, it was realized that these misposition the intercadherin domain boundary, and consequently these constructs lacked three amino acids of the first cadherin domain. Addition of these amino acids, together with the intercadherin domain linker sequence, enabled phosphorylation of a Ds3 single-cadherin domain construct (Ishikawa, 2008).

A weak similarity between Fj and the bacterial kinase HipA, and between Fj and the mammalian lipid kinase phosphatidylinositol 4-kinase II (PI4KII), has been suggested previously on the basis of bioinformatic analyses in which HipA or PI4KII were used as the starting point for PSI-BLAST searches. Asp residues play critical roles in catalysis and in the coordination of Mg²⁺ in these and other kinases, and the loss of Fj kinase activity associated with mutation of the conserved DNE motif is thus consistent with the inference that Fj is related to other kinases. A single Fj ortholog, Fjx1, is present in a range of vertebrate species, including humans (Ishikawa, 2008).

To investigate the biological requirement for Fj kinase activity, the catalytically inactive fj^GGG mutant was assayed in vivo. A V5 epitope-tagged form of this gene was expressed in transgenic Drosophila. At the same time, V5-tagged wild-type fj was contructed. To ensure that both forms were expressed in similar amounts, site-specific integration was used to insert transgenes at the same chromosomal location. Immunostaining confirmed that Fj^GGG:V5 and Fj:V5 both exhibited normal Golgi localization and were expressed in similar amounts. Uniform overexpression of fj reduces the growth of legs and wings and interferes with normal PCP. Fj:V5 exhibited phenotypes consistent with previous studies, but Fj^GGG:V5 was completely inactive. Thus, mutation of the DNE motif in Fj abolishes its biological activity (Ishikawa, 2008).

The identification of Fj's cadherin domain kinase activity provides a biochemical explanation for the influence of Fj on Fat signaling and supports a model in which Fj directly phosphorylates Fat and Ds as they transit through the Golgi to influence their activity, presumably by modulating interactions between their cadherin domains. Because there was a substantial difference in the efficiency with which individual cadherin domains could be modified by Fj, both in cell-based and in vitro assays, it is also possible that differences in the extent of Fat and Ds phosphorylation normally occur in vivo and might differentially modify their binding or activity (Ishikawa, 2008).

DEVELOPMENTAL BIOLOGY

Embryonic

The first dachsous transcripts are detectable during gastrulation in a pair-rule pattern of six weak epidermal stripes and in a prominent stripe at the amnioproctodeal invagination. During germ-band extension, DS mRNA accumulates in a segmentally repeated pattern of 14 stripes and in the procephalon. The most pronounced expression of ds is observed during the extended germ-band stage, mainly in the forming tracheal pits. At the beginning of head involution, DS mRNA appears in the anterior spiracles and again in stripes of the segmental groves and buds, while it remains weakly expressed in the remnants of the tracheal pits. DS mRNA is first detected in the primoridal leg discs,which form in the ventral posterior part of each throracic segment. At late stage 14, ds is expressed strongly in the nearly fused labial buds and at invaginations of the maxillary segment while it continues to be expressed in the leg disc primordium, along the segmental folds, and probably in the apodemes (muscle attachment sites of the ectoderm). After dorsal closure, expression persists only in the apodemes and in the head region (Clark, 1995).

Larval

In third-instar larvae, ds transcripts are found in the imaginal discs and the brain. In the supraesophageal ganglion (brain), ds is expressed in two areas of the areas of the optic lobe and in a region that might belong to the mushroom body. In imaginal discs, strong ds expression occurs frequently along folds separating the anlagen of distinct imaginal structures. In the antennal disc, ds is expressed in the arista and first and second antennal segment anlagen; in the eye disc, ds transcripts are abundant along the folds of the future bristle region of the head capsule. Expression of ds is also observed in humeral, genital and labial discs. In leg discs, ds is expressed strongly in the anlagen of the tarsal joints and in particular, the most proximal leg segment. Similarly, ds expression is strongest in the pleural, dorsal, hinge, and prescutal regions of the wing disc, whereas the anlage of the future wing blade is virtually free of DS mRNA. In contrast, the haltere disc exhibits high levels of DS transcripts in the capitellum, pedicle, and scabellum, while expression in the notum remains relatively low (Clark,1995).

Homothorax and Extradenticle are expressed in the proximal domain of the leg (the Hth domain) : all the other factors studied (Wingless, Decapentaplegic and Distalless) are expressed in more distal regions (the Dll domain). Dachsund (Dac) is expressed in an intermediate domain, dorsal and lateral to the more distal Dll domain (the Dac domain). What follows is a more complete description of these domains. The expression of several targets of the signaling molecules Wg and Dpp were examined in relation to the hth expression domain. dpp expression in the leg disc at the early third larval instar stage consists of a sector that originates at the center of the disc, extends dorsally to the periphery and shows extensive overlap with Hth. omb, a target of the Dpp-signaling pathway, is expressed in a dorsal sector that, in contrast to dpp, extends dorsally only to abut, but not overlap with, the hth domain. wg expression consists of a ventral sector of cells that extends from the center to the periphery of the disc, whereas H15, an enhancer trap line that requires wg signaling for its activation, is largely not transcribed in the hth domain. The restriction of these Wg and Dpp target genes to non-hth-expressing cells suggests that hth restricts signaling by these two molecules. By the late third larval instar stage, there is a small degree of overlap between hth and omb expression as well as between hth and H15. This expression corresponds to the trochanter domain where gene activation can occur independent of the Wg- and Dpp-signaling pathways. Unlike omb and H15, the Dll and dac genes require input from both the Dpp and Wg signal transduction pathways to be activated in leg discs. Dll encodes a homeodomain protein present in the central portion of leg discs, and its activation requires the highest concentrations of Wg and Dpp. dac encodes a nuclear protein and a putative transcription factor whose expression is repressed by high concentrations, and activated by intermediate concentrations, of Wg and Dpp. By performing triple stains for the dac^P-lacZ reporter gene, and Dll and Hth proteins at the early third larval instar stage, it was found that the leg disc is defined by three non-overlapping domains of gene expression. The distal-most domain of the leg disc contains Dll protein (the Dll domain). Dorsal and dorsolateral, but not ventral, to the Dll domain are cells that express dac (the Dac domain). The proximal-most cells of the disc, which surround the dac and Dll domains, express hth (the Hth domain). At the mid 3rd larval instar stage (~96 hours after egg lay, or AEL), the distal-most cells express only Dll and are surrounded by a ring of cells that express both Dll and dac. At this stage, there is also a dorsal patch of cells that express dac but not Dll. hth expression remains limited to the proximal-most cells of the disc and shows no overlap with dac or Dll. By the late 3rd larval instar stage (~120 hours AEL), hth is still not co-expressed with dac or Dll, with the exception of a thin band of cells corresponding to the trochanter domain, where all three genes are co-expressed. Gene expression in the trochanter domain is likely to represent secondary patterning events, because it is not dependent on Wg- or Dpp-signaling. At this stage dac expression also surrounds and partially overlaps the Dll expression domain. It is proposed that the Dll and Dac domains, where hth transcription is off and Exd is cytoplasmic, are Dpp- and/or Wg-responsive domains, as demonstrated by the ability of these cells to respond to these signals by activating the target genes Dll, dac, omb and H15. In contrast, the hth domain, where hth is active and Exd is nuclear, is a Wg- and/or Dpp-non- responsive domain, where these signals are present but cannot activate these targets (Abu-Shaar, 1998).

The integument of the Drosophila adult abdomen bears oriented hairs and bristles that indicate the planar polarity of the epidermal cells. Four polarity genes, frizzled (fz), prickle (pk), Van gogh/strabismus (Vang/stbm) and starry night/flamingo (stan/fmi) were examined in this study, and what happens when these genes are either removed or overexpressed in clones of cells was examined. The edges of the clones are interfaces between cells that carry different amounts of gene products, interfaces that can cause reversals of planar polarity in the clone and wild-type cells outside them. To explain, a model is presented that builds on an earlier picture of a gradient of X, the vector of which specifies planar polarity and depends on two cadherin proteins, Dachsous and Fat. It is conjectured that the X gradient is read out, cell by cell, as a scalar value of Fz activity, and that Pk acts in this process, possibly to determine the sign of the Fz activity gradient (Lawrence, 2004).

It has been proposed that, in the abdomen of Drosophila, morphogen gradients (Hh in the A compartment and Wg in the P compartment) organise a secondary gradient ('X'); the vector of X specifying the polarity of each cell. Although the composition of X is unknown, at least three proteins, Fj, Ds and Ft, are implicated. All three may be expressed, or be active, in bell-shaped distributions that peak near the A/P (Ds) or P/A (Fj, Ft) boundaries. Ds and Ft are transmembrane proteins in the cadherin superfamily; Fj probably acts in the Golgi. Ds and Ft are integrated into the membrane, suggesting that the X gradient itself may not be diffusible but instead might depend on information transfer from cell to cell (Lawrence, 2004).

How does Hh set up the X gradient? Although changing the real or perceived level of Hh does affect polarity, many clones (for example clones that lack Smo, an essential component of Hh reception) show there is no simple correlation between Hh concentration and polarity. For instance, large smo^- clones in the center of the A compartment are polarised normally, even though they are blind to Hh. Also, while smo^- clones in some regions of the A compartment do affect polarity, both mutant and wild-type cells are repolarised. Both these observations argue for some transfer of information about polarity between cells, a process that would be at least partly Hh independent. This process involves four genes (stan, fz, Vang and pk) that probably act downstream of ds, ft and fj (Lawrence, 2004).

dachsous is required for Wg-dependent pattern formation in the Drosophila wing disc

The dachsous (ds) gene encodes a member of the cadherin family involved in the non-canonical Wnt signaling pathway that controls the establishment of planar cell polarity (PCP) in Drosophila. ds is the only known cadherin gene in Drosophila with a restricted spatial pattern of expression in imaginal discs from early stages of larval development. In the wing disc, ds is first expressed distally, and later is restricted to the hinge and lateral regions of the notum. Flies homozygous for strong ds hypomorphic alleles display previously uncharacterized phenotypes consisting of a reduction of the hinge territory and an ectopic notum. These phenotypes resemble those caused by reduction of Wingless during early wing disc development. An increase in Wg activity can rescue these phenotypes, indicating that Ds is required for efficient Wg signaling. This is further supported by genetic interactions between ds and several components of the Wg pathway in another developmental context. Ds and Wg show a complementary pattern of expression in early wing discs, suggesting that Ds acts in Wg-receiving cells. These results thus provide the first evidence for a more general role of Ds in Wnt signaling during imaginal development, not only affecting cell polarization but also modulating the response to Wg during the subdivision of the wing disc along its proximodistal (PD) axis (Rodríguez, 2004).

In second instar larvae, ds-lacZ expression is essentially confined to the distal part of the wing disc, but is absent in those distal A cells in which Wg strongly accumulates. This Wg expression constitutes the earliest marker for the nascent wing pouch. Soon thereafter, when Wg expression is expanded to the adjacent P cells, ds-lacZ expression fades away and becomes confined to a ring of cells around the prospective wing pouch. At this stage, most of the hinge cells located between the prospective notum and wing pouch express ds-lacZ at high levels, as revealed by the Iro and Nub markers. A weak expression of ds-lacZ overlaps with the periphery of the Nub domain and marks the region that will become the proximal wing. At third instar, ds-lacZ expression is also observed within the lateral regions of the prospective notum. An antibody directed against the cytoplasmic region of Ds protein reveals a Ds protein distribution similar to the ds-lacZ expression pattern and an apical location at the plasma membrane. From these results, it is concluded that ds-lacZ expression is one of the earliest and most specific markers of the prospective hinge during the second and early third larval instar (Rodríguez, 2004).

During patterning and growth of the wing blade, Wg distribution has been proposed to signal to distant cells in a concentration-dependent manner. Several mechanisms, such as the interaction of Wg with heparin sulfate-containing proteoglycans, as well as regulated endo- and exocytosis, are involved in shaping the gradient and delimiting the range of signaling. Wg protein is predominantly located at the apical surface in the producing cells, and in the embryo it has been demonstrated that this sub-cellular location is essential for its signaling activity. When the hinge territory is already specified at early third instar, wg is activated in these cells and acts as a cell proliferation signal necessary for the development of most structures. Wg expression within the hinge can be described as two rings, the 'inner ringí (IR) and the 'outer ringí (OR), which overlap with the areas of low (ring I) and high expression of ds-lacZ (ring II), respectively. In this context, since Ds is also apically located, it was of interest to determine whether Ds has a role in Wg-producing cells. To test this, the distribution of Wg was examined in large clones of ds mutant cells. Two results were obtained from these experiments: (1) the level of Wg in the producing cells was slightly increased with respect to neighbouring wild-type cells, and (2) the Wg gradient within mutant tissue appears to be broader. These results should imply a higher signaling capacity of Wg in ds mutant cells; however, that was not the case. In ds clones, Wg accumulation was less marked apically and was relatively more abundant in the baso-lateral region than in the wild-type cells. Interestingly, this phenomenon seems not to be strictly cell autonomous, because adjacent wild-type cells also displayed a similar abnormal sub-cellular localization of Wg protein. This effect could be due to basal Wg protein diffusing more rapidly to adjacent cells than apical protein does, as has been observed in the embryo. Taken together, these observations suggest that Ds protein contributes to the apical localization of Wg protein at the plasma membrane. It is thought unlikely that this function of Ds is responsible for the early PD patterning defects in DNW discs, since ds and wg are expressed in complementary domains during early larval development (Rodríguez, 2004).

Whether Ds is required for Wg-mediated patterning in imaginal discs other than the wing disc was examined by analyzing the genetic interactions between ds and several components of the Wg pathway during leg development. Homo- and hetero-allelic combinations of ds cause a reduction of the segment size and fusion of the tarsal segments, with partial elimination of the tarsal joints. This phenotype resembles some defects associated with the loss of function of pangolin/dTCF, and legless/BCL9. Therefore, the levels of Wg signaling were manipulated in mid-strength heteroallelic combinations of ds. The loss of one wild-type copy of dsh enhanced the fusion of leg tarsi and shortened the leg segments. By contrast, the leg phenotype of ds showed a complete recovery of the tarsal joints and an increase in the length of the segments when one dose of the nkd gene, an antagonist of the Wg pathway, was eliminated. Taken together, these findings support a more general role for ds in Wg-mediated patterning processes (Rodríguez, 2004).

The wing primordium is specified as a few anterior cells that express wg at the distal-most part of the wing imaginal disc at second larval instar. Slightly later, wg is also expressed in P cells and these cells are recruited into the wing fate. In 'double-notum-winglet' (DNW) ds strongly hypomorphic mutant discs, the level of Ds protein is highly reduced and only the initial anterior group of Wg-expressing cells becomes specified into the wing fate. The levels of this initial Wg expression seems not to be affected in DNW discs. However, neither the P cells abutting the initial anterior Wg domain nor the surrounding cells of this early wing primordium are able to respond to Wg, leading to the formation of a wing pouch composed exclusively by A cells. Moreover, the activation of Wg target genes (such as hth, required for the specification of hinge cells) fails in DNW discs, and, consequently, the proximal wing and hinge structures do not develop. The significantly reduced rings of ds-lacZ, hth and zfh2 expression in DNW discs most likely reflect the residual Ds activity retained in the ds^38k mutant. Cells close to the Wg source might thus still be able to respond to high Wg levels during early stages of wing development. However, under null conditions for ds (ds^D36) the expression of zfh2 is eliminated (Rodríguez, 2004).

Thus, in addition to its function in PCP, ds plays a role in early patterning when the specification of the different territories along the PD axis takes place in response to Wg. Initially, ds facilitates the recruitment of P cells into the wing fate in response to Wg. Subsequently, Ds promotes the activation of Wg target genes in the surrounding cells to specify the hinge. Note that once the hinge cells have been specified in response to Wg signaling, ds seems to be dispensable for global wing disc patterning, as the ‘classical’ ds^38k phenotype shows. In this case, only mild defects such as slight tissue overgrowth or polarity defects were observed, suggesting additional functions of ds related to cell adhesion (Rodríguez, 2004).

Ectopic expression of Dpp in wing cells of DNW discs restores both the formation of the AP border and cell proliferation within the wing pouch, indicating that both Wg and Dpp orchestrate these events. Only cells previously committed to the wing fate by Wg are able to proliferate in response to Dpp, as the UAS-dpp/dpp-Gal4 and UAS-dpp/omb-Gal4 experiments suggest. In the ds mutant background, omb is expressed in anterior wing cells, albeit in the absence of the AP border/Dpp source within the wing pouch, suggesting that this initial omb expression might not be Dpp dependent. Similar results were observed for spalt (sal), another known target gene of dpp. It is proposed that Ds primarily regulates Wg signaling in the initial recruitment of P cells into putative wing territory. Once this initial recruitment has occurred, Dpp expression is established and Dpp signaling can contribute to the further recruitment of P cells. Expression of UAS-dpp in anterior wing pouch cells of ds mutant discs using omb-Gal4 can bypass the initial requirements for Wg in P cell recruitment, leading to the observed wing pouch rescue (Rodríguez, 2004).

In vertebrates, during telencephalon formation, the organization into different structures requires the expression of different cadherins in adjacent regions to maintain a compartment boundary based on differential cell affinity features. It has been suggested that the expression pattern of each of these cadherins is under the control of specific signaling cascades (Rodríguez, 2004).

In Drosophila, during imaginal disc development, indirect evidence has suggested that cell adhesion might be under the control of the same signaling pathways that control cell proliferation and patterning. The smooth borders of clones mutant for thick vein (tkv), the receptor of Dpp, or smoothened (smo), a downstream component of the Hedgehog (Hh) signaling pathway, indicate that mutant cells change their affinity properties and therefore try to minimise the contact with surrounding wild-type cells. Nevertheless, little is known about the molecules involved in these adhesiveness differences. Recent work has proposed that both tartan and capricious (caps), two transmembrane proteins regulated by ap, are putative candidates to maintain the affinity boundary between dorsal and ventral cells. However, whereas clones ectopically expressing tartan and caps in V cells tend to contact D cells, the elimination of tartan and caps in clones from D cells had no effect on DV boundary formation (Rodríguez, 2004).

In the DNW phenotype, the ectopic notum develops from cells of the hinge territory. According to the proposed subdivision into concentric rings (I to III), cells from the outermost ring III expressing Tsh and Ds will give rise to that part of the body wall that is excluded from the notum region. In DNW discs, the absence of Ds produces an expansion of notal-specific iro-C expression to more distal positions to fill up the Tsh domain. These distal cells acquire a notum fate, generating an ectopic notum similar to wg¹ mutant flies (Rodríguez, 2004).

Thus, Ds protein contributes to hinge/notum boundary formation by means of an affinity border. This process would occur at early second instar when Iro-C expression is capable of specifying the notum fate. This finding provides the first evidence that a cadherin is able to maintain the cell boundary between two adjacent territories in Drosophila (Rodríguez, 2004).

How does ds participate in Wg signaling? Several findings point out a specific role of Ds in the modulation of Wg signaling: (1) the elimination of zfh2 expression in ds mutant clones; (2) the genetic interactions of ds alleles with several components of the Wg signaling pathway, and (3) the rescue of the DNW phenotype by increasing Wg levels. It has been shown that Ds is associated with adherens junctions at the apical surface of the imaginal cells, to mediate cell-cell adhesion. A major step of the cell adhesion mechanism requires interaction of the cytoplasmic tail with Arm/ß-catenin to connect the cadherin-catenin complex to the actin cytoskeleton. Thus, the phenotype could reflect changes in the balance between cytoplasmic Arm versus Arm anchored to the plasma membrane. If this were the case, then a reduction of ds function would increase Wg signaling; however, the results presented above indicate that loss of ds decreases Wg signaling. Moreover, sequence analysis has shown that the ß-catenin binding motifs in the Ds protein, which have to be in tandem to be functional, are separated by a stretch of amino acids, further discarding the possibility that Ds binds directly to Arm to modulate its cytoplasmic levels (Rodríguez, 2004).

Alternatively, the apical plasma membrane acts as a structural center that contains crucial components that modulate the Wg pathway, such as Dsh, E-APC and Axin. Axin and E-APC, promote the degradation of cytoplasmic Arm, the main effector of the Wg cascade. Previous work has shown that, upon binding of Wg in the receiving cells, the Axin/E-APC complex becomes anchored to the plasma membrane to prevent Arm degradation. In this context, Ds protein, as part of the adherens junctions, could be the cadherin required to anchor this degradation complex to the plasma membrane. In ds mutant cells, the cytoplasmic levels of the Axin/E-APC complex would be higher and, therefore, Wg signaling would decrease. In agreement with this hypothesis, mild ds phenotypes are enhanced when a copy of dsh gene is eliminated. Still, Ds could act at the level of Wg reception, by increasing the Fz/Wg-binding affinity or by recruiting Fz molecules to the apical plasma membrane, as has been demonstrated for the cadherin Fmi in the PCP processes (Rodríguez, 2004).

Early anterior Wg activity initiates specification of the PD axis in the wing disc: To date, the current model explaining the specification of the territories along the PD axis assumes that the initial anterior Wg expression at second instar is required only for cells to acquire the wing fate. It is only later, when wg is expressed in two concentric rings that its function is required to specify the hinge territory (Rodríguez, 2004).

Wg has been shown to be required for the development of the hinge. In contrast, Wg activates downstream genes such as hth or zfh2 to specify the hinge fate. In contrast, Wg controls cell proliferation when it is expressed from early third instar into the IR and OR rings. It has been established that the specification of the hinge takes place later than the wing; however, the data show that an early and timely limited depletion of Wg activity causes a failure in hinge specification. This is mainly based on the observation that only early-induced ds clones abolish zfh2 expression required for hinge formation. In ds mutant clones induced later, hinge development is unaffected, although a perdurance of ds activity in these clones cannot be excluded. Still, the rescue of hinge development in DNW discs that ectopically express Wg under dpp-Gal4 further support an early specification of the hinge. In these discs, ectopic Wg expression stays confined to the AP border. At early stages, the AP border must be located close enough to the nascent wing primordial to allow the spreading of Wg into regions destined to become hinge territory. At late stages, the narrow stripe of ectopic Wg expression can no longer account for the maintenance of the whole hinge territory. It is rather the Wg within the IR and OR that maintains hth expression and, with it, the specification of the hinge fate. At this stage, either Wg works independently of ds or its requirements for ds are lower. Thus, if hinge specification is not initiated early upon ds and wg activities, wg expression cannot be established and the development of the hinge is aborted (Rodríguez, 2004).

The present results provide insights that help in the understanding of how the PD axis is established in the wing disc. The initial event in this process would be the early activity of Wg. When Wg is expressed at the distal part of the wing disc in a small group of anterior cells, it not only promotes the activation of target genes like vg, nub or scalloped (sd) in the wing cells, but also the expression of hth and zfh2 to specify the hinge. At the same time, Wg would repress tsh or vein (vn) at the distal part of the wing disc to separate the proximal wing and hinge regions from the body wall where Egfr signaling activates notum-specific genes like iro-C. Thus, in cooperation with dpp, wg establishes the AP and PD axis in the prospective wing and hinge regions (Rodríguez, 2004).

In DNW discs, even though the Dpp source is distantly and asymmetrically located with respect to the wing pouch, anterior wing cells differentiate into distinct cell types in a mirror image disposition. This result suggests that specific positional information might be provided independently of dpp. Ap in combination with Wg might contribute to this initial AP positional information. Once P cells are recruited into the wing fate, Dpp takes over and promotes pattern formation along the AP axis, as well as proliferation within the wing pouch (Rodríguez, 2004).

Action of fat, four-jointed, dachsous and dachs in distal-to-proximal wing signaling

In the Drosophila wing, distal cells signal to proximal cells to induce the expression of Wingless, but the basis for this distal-to-proximal signaling is unknown. Three genes that act together during the establishment of tissue polarity, fat, four-jointed and dachsous, also influence the expression of Wingless in the proximal wing. fat is required cell autonomously by proximal wing cells to repress Wingless expression, and misexpression of Wingless contributes to proximal wing overgrowth in fat mutant discs. Four-jointed and Dachsous can influence Wingless expression and Fat localization non-autonomously, consistent with the suggestion that they influence signaling to Fat-expressing cells. dachs is identified as a gene that is genetically required downstream of fat, both for its effects on imaginal disc growth and for the expression of Wingless in the proximal wing. These observations provide important support for the emerging view that Four-jointed, Dachsous and Fat function in an intercellular signaling pathway, identify a normal role for these proteins in signaling interactions that regulate growth and patterning of the proximal wing, and identify Dachs as a candidate downstream effector of a Fat signaling pathway (Cho, 2004).

There is a progressive elaboration of patterning along the PD axis over the course of wing development. During the second larval instar, interactions among the Epidermal Growth Factor Receptor, Dpp and Wg signaling pathways divide the wing disc into a dorsal region, which will give rise to notum, and a ventral region, from which the wing will arise. An initial PD subdivision of the wing is then effected by signaling from the AP and DV compartment boundaries, which promotes the expression of two genes, scalloped and vestigial, that encode subunits of a heterodimeric transcription factor (Sd-Vg) in the center of the wing. This subdivides the wing into distal cells, which give rise to the wing blade, and surrounding cells, which give rise to proximal wing and wing hinge structures. The proximal wing is further subdivided into a series of molecularly distinct domains. Studies of Sd-Vg function in the wing led to the realization that the elaboration of this finer pattern depends in part upon signaling from the distal, Sd-Vg-expressing cells, to more proximal cells. Thus, mutation of vg leads to elimination, not only of the wing blade, where Vg is expressed, but also of more proximal tissue. Conversely, ectopic expression of Vg in the proximal wing reorganizes the patterning of surrounding cells (Cho, 2004 and references therein).

A key target of the distal signal is Wg, which during early third instar is expressed in a ring of cells that surround the SD-VG-expressing cells, and which later becomes expressed in a second, more proximal ring. Wg expression in the inner, distal ring within the proximal wing is regulated by an enhancer called spade-flag (spd-fg), after an allele of wg in which this enhancer is deleted (Neumann, 1996). Studies of this allele, together with ectopic expression experiments, have revealed that Wg is necessary and sufficient to promote growth of the proximal wing. Wg also plays a role in proximal wing patterning; it acts in a positive-feedback loop to maintain expression of Homothorax (Hth). The rotund (rn) gene has been identified as an additional target of distal signaling (Cho, 2004 and references therein).

This work identified Four-jointed (Fj), Dachsous (Ds), Fat and Dachs as proteins that influence signaling to proximal wing cells to regulate Wg and rn expression. Fj is a type II transmembrane protein, which is largely restricted to the Golgi. Null mutations in fj do not cause any obvious defects in the proximal wing. However, fj plays a role in the regulation of tissue polarity, yet acts redundantly with some other factor(s) in this process. Mutations in fat or ds can also influence tissue polarity. Although the molecular relationships among these proteins are not well understood, genetic studies suggest that fj and ds act via effects on fat, and both fj and ds can influence Fat localization in genetic mosaics (Cho, 2004 and references therein).

Interestingly, alleles of fj, ds and fat, as well as alleles of another gene, dachs, can result in similar defects in wing blade and leg growth. The similar requirements for these genes during both appendage growth and tissue polarity, together with the expression patterns of fj and ds in the developing wing, led to this investigation of their requirements for proximal wing development. All four genes influence the expression of Wg in the proximal wing, and genetic experiments suggest a pathway in which Fj and Ds act to modulate the activity of Fat, which then regulates transcription via a pathway that includes Dachs. These observations lend strong support to the hypothesis that Fj, Ds and Fat function as components of an intercellular signal transduction pathway, implicate Dachs as a key downstream component of this pathway, and identify a normal role for these genes in proximodistal patterning during Drosophila wing development (Cho, 2004).

The common feature of all of the manipulations of FJ and DS expression carried out in this study is that Wg expression, and by inference, Fat activity, can be altered when cells with different levels of Fj or Ds are juxtaposed. In the case of Fj, its normal expression pattern, and effects of mutant and ectopic expression clones are all consistent with the interpretation that juxtaposition of cells with different levels of Fj is associated with inhibition of Fat in the cells with less Fj and activation of Fat in the cells with more Fj. The influence of Ds, however, is more variable. Studies of tissue polarity in the eye suggest that Ds inhibits Fat activity in Ds-expressing cells, and/or promotes Fat activity in neighboring cells. The predominant effect of Ds during early wing development is consistent with this, but its effects in late discs are not. Studies of tissue polarity in the abdomen suggest that the Ds gradient might be interpreted differently by anterior versus posterior cells, and it is possible that a similar phenomena causes the effects of Ds to vary during wing development (Cho, 2004).

The influence of ds mutation on gene expression and growth in the wing is much weaker than that of fat. It has been suggested that Fj might influence Fat via effects on Ds, and fj mutant clones have been observed to influence Ds protein staining. The observations are consistent with the inference that both Ds and Fj can regulate Fat activity, but they do not directly address the question of whether Fj acts through Ds. They do, however, indicate that even the combined effects of Fj and Ds cannot account for FAT regulation, and, assuming that the strongest available alleles are null, other regulators of Fat activity must exist. It is presumably because of the counteracting influence of these other regulators that alterations in Fj and Ds expression have relatively weak effects. In addition, according to the hypothesis that Fat activity is influenced by relative rather than absolute levels of its regulators, the effects of Fj or Ds could be expected to vary depending upon their temporal and spatial profiles of expression, as well as on the precise shape and location of clones (Cho, 2004).

The observations imply the existence of at least two intracellular branches of the Fat signaling pathway. One branch involves the transcriptional repressor Grunge, influences tissue polarity, certain aspects of cell affinity, and fj expression, but does not influence growth or wg expression. An alternative branch does not require Grunge, but does require Dachs. Dachs is implicated as a downstream component of the Fat pathway, based on its cell autonomous influence on Fat-dependent processes, and by genetic epistasis. The determination that it encodes an unconventional myosin, and hence presumably a cytoplasmic protein, is consistent with this possibility. It also suggests that Dachs does not itself function as a transcription factor, and hence implies the existence of other components of this branch of the Fat pathway. This Grunge-independent branch influences Wg expression in the proximal wing and imaginal disc growth. However, further studies will be required to determine whether Dachs functions solely in Grunge-independent Fat signaling, or whether instead Dachs is required for all Fat signaling (Cho, 2004).

The observations that fj expression is regulated by Sd-Vg, and that fj is both necessary and sufficient to modulate the distal ring of Wg expression in the proximal wing, suggest that Fj influences the activity of a distal signal, which then acts to influence Fat activity. However, the relatively weak effects of fj indicate that other factors must also contribute to distal signaling, just as fj functions redundantly with other factors to influence tissue polarity. Since Ds expression is downregulated in a domain that is broader than the Vg expression domain, a direct influence of Vg on the Ds gradient is unlikely, and the essentially normal appearance of Wg expression in the proximal wing in fj ds double mutants implies that Ds is not a good candidate for the hypothetic factor Signal X. Rather, it is suggested that Ds acts in parallel to signaling from Vg-expressing cells to modulate Fat activity. This Vg-independent effect would account for the remnant of the distal ring that sometimes appears in vg null mutants. Importantly though, the observation that the phenotypes of hypomorphic dachs mutant clones on Wg expression are more severe than fj and ds suggests that the hypothesized additional factors also act via the Fat pathway. It is also noted that the limitation of Wg expression to the proximal wing even in fat mutant clones implies that Wg expression both requires Nubbin, and is actively repressed by distally-expressed genes (Cho, 2004).

The recovery of normal Wg expression by later stages in both fj and dachs mutant clones implies that the maintenance of Wg occurs by a distinct mechanism. Prior studies have identified a positive-feedback loop between Wg and Hth that is required to maintain their expression. It is suggested that once this feedback loop is initiated, Fat signaling is no longer required for Wg expression. Moreover, the recovery of normal levels of Wg at late stages suggests that this positive-feedback loop can amplify reduced levels of wg to near normal levels (Cho, 2004).

The distinct consequences of Vg expression and Fj expression in clones in the proximal wing suggest that another signal or signals, which are qualitatively distinct from the Fj-dependent signal, is also released from VG-expressing cells. When Vg is ectopically expressed, Wg is often induced in a ring of expression that completely encircles it. However, this is not the case for Fj-expressing clones. Both Vg- and Fj-expressing clones can activate rn and wg only within NUB-expressing cells, but Vg expression can result in non-autonomous expansion of the Nub domain, and this expansion presumably facilitates the expression of Wg by surrounding cells. Another striking difference between Vg- and Fj-expressing clones is that in the case of ectopic Fj, enhanced Wg expression is only in adjacent cells. By contrast, in the case of Vg, Wg expression initiates in neighboring cells, but often moves several cells away as the disc grows, resulting in a gap between Vg and Wg expression. This gap suggests that a repressor of Wg expression becomes expressed there, and recent studies have identified Defective proventriculus (Dve) as such a repressor (Cho, 2004).

In strong fat mutants, the wing discs become enlarged and have extra folds and outgrowths in the proximal wing. The disproportionate overgrowth of the proximal wing is due to upregulation of Wg in this region, as demonstrated by its suppression by wg^spd-fg. At the same time, clones of cells mutant for fat overgrow in other imaginal cells, and fat wg^spd-fg discs are still enlarged compared with wild-type discs. Thus, Fat appears to act both by regulating the expression of other signaling pathways (e.g. Wg), and via its own, novel growth pathway. The identification of additional components of this pathway will offer new approaches for investigating its profound influence on disc growth (Cho, 2004).

Effects of Mutation or Deletion

The adult phenotype of ds mutants is consistent with the high levels of ds transcript in imaginal discs. In all known alleles defects are seen with 100% penetrance in the legs, wings, and thorax. In contrast, eye defects, apparent as rough patches, occur at a low frequency. On ds wings, the anterior cross-vein is displaced distally, that is, closer to the posterior cross-vein; the legs are stubby, with a reduced number of tarsal joints in stronger alleles, and the thorax is broadened. These adult phenotypes are more pronounced in stronger mutant alleles, with the addition of duplicate bristles on the notum and wings stiffly held out, with broken and ectopic cross-veins. All ds mutant discs exhibit normal size range with no overgrowth. Therefore, in contrast to mutations in the fat gene, encoding a similar cadherin protein, ds mutations appear to alter imaginal disc morphogenesis exclusively without affecting cell proliferation (Clark, 1995).

The adult cuticular wing of Drosophila is covered by an array of distally pointing hairs that reveal the planar polarity of the wing. Mutations in dachsous disrupt this regular pattern, and do so by affecting frizzled signaling. Mutations in dachsous also result in a tissue polarity phenotype that at the cellular level is similar to frizzled, dishevelled and prickle, as many cells form a single hair of abnormal polarity. Although their 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 and suggests that in a dachsous mutant the frizzled pathway is functioning - but in an abnormal way. Genetic experiments indicate 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 it has been found that frizzled clones in a dachsous mutant background display enhanced domineering non-autonomy, and that the anatomical direction of this domineering non-autonomy is altered in regions of dachsous wings that have 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 (Adler, 1998).

Recessive lethal mutations of the lethal(2)giant discs (l(2)gd) and lethal(2)fat (l(2)ft) loci of Drosophila melanogaster cause imaginal disc hyperplasia during a prolonged larval stage. Imaginal discs from l(2)ft l(2)gd or Gl(2)gd double homozygotes show more extensive overgrowth than in either single homozygote, and double homozygous l(2)ft l(2)gd mitotic clones in adult flies show much more overgrowth than is seen in clones homozygous for either l(2)gd or l(2)ft alone. dachsous (ds) also acts as an enhancer of l(2)gd, producing dramatically overgrown discs and causing failure to pupariate in double homozygotes. The comb gap (cg) mutation, which also interacts with ds, greatly enhances the tendency of imaginal discs from l(2)gd larvae to duplicate as they overgrow. If l(2)gd homozygotes are made heterozygous for l(2)ft, then several discs duplicate, indicating that l(2)ft acts an a dominant enhancer of l(2)gd. l(2)ft also acts as a dominant enhancer of l(2)gd, and conversely l(2)gd acts as a dominant modifier of l(2)ft. The enhancement of overgrowth caused by various mutant combinations is accompanied by changes in expression of Decapentaplegic and Wingless. These results show that tumor suppressor genes act in combination to control cell proliferation, and that tissue hyperplasia can be associated with ectopic expression of genes involved in pattern formation (Buratovich, 1997).

Two viable fly stocks have been generated by altering the level of Armadillo available for signaling. Flies from one stock overexpress Armadillo (Arm^over) and, as a result, have increased vein material and bristles in the wings. Flies from the other stock have reduced cytoplasmic Armadillo following overexpression of the intracellular domain of DE-cadherin (Arm^under). These flies display a wing-notching phenotype typical of wingless mutations. Both misexpression phenotypes can be dominantly modified by removing one copy of genes known to encode members of the wingless pathway. This paper identifies and describes further mutations that dominantly modify the Armadillo misexpression phenotypes. These mutations are in genes encoding three different functions: establishment and maintenance of adherens junctions, cell cycle control, and Egfr signaling (Greaves, 1999).

Mutations in 17 genes (26 deficiencies) were characterized that interact with Arm^over and/or Arm^under. Interaction strength varies from deficiency to point mutation, suggesting that several genes in the original deficiencies could have contributed to, or modified, the interaction. Only for 7 of the 17 genes have interactions been identical between the point mutation and the corresponding starting deficiency. The 17 genes were sorted into four groups. Group 2 consists of genes required for cell adhesion: This group includes shotgun (which encodes DE-cadherin), as expected. Also uncovered were fat (ft) and dachsous (ds). These two genes encode nonclassical cadherin characterized by a huge extracellular domain containing up to 35 cadherin repeats and a bipartite Arm binding site. Interactions with these two mutants are similar to those observed with shotgun (DE-cadherin), the only difference being that ft interacts more weakly than shg with Arm^over. In addition to genes encoding cadherins (classical and nonclassical), interactions have been observed with some of the genes known to be essential (directly or indirectly) for the assembly or maintenance of adherens junctions—stardust (sdt), discs-large (dlg), and crumbs (crb). These interact in the same direction as shg; however, the suppression of Arm^under is always weaker and only dlg^M52 enhances Arm^over to the same extent as zw3^M11 (Greaves, 1999).

The interaction with shotgun (encoding DE-cadherin) itself is not very illuminating since it is expected that the phenotype caused by an excess of intracellular cadherin domain will be suppressed by decreasing endogenous cadherin levels. Still, this interaction shows that the level of overexpression afforded by the Gal4p system is within physiological levels. Interaction with fat and dachsous suggests that these two nonclassical cadherins interact (maybe directly) with Arm. Initial analysis of the intracellular domain of Fat and Dachsous fail to identify an Arm/ß-catenin binding site homologous to that found in E-cadherin. However, subsequent sequence examination suggests the existence of a bipartite site. Genetic interactions with fat and dachsous strongly suggest that this proposed site is functional, and thus removing one copy of the fat or dachsous gene would release additional Arm to the cytoplasm and make it available for use in Wg transduction. Interactions with fat and dachsous in the eye confirm the ability of these genes to modify cytoplasmic Arm levels. It also indicates that these genes are expressed in the eye and may be functional there (Greaves, 1999).

Cadherin-N (CadN) binds to Arm. Therefore the failure of CadN to interact in this screen suggests that CadN may not be expressed to significant levels in the posterior compartment of wing imaginal discs or in eye precursors. In contrast, crumbs (crb) and stardust (sdt) do interact. The proteins encoded by these genes are not thought to participate in junctional complexes per se. Rather, they control the biogenesis of the junctions. It is suggested that decreasing the activity of crb or sdt has a quantitative effect on the number or size of adherens junctions and this would lead to more Arm being released from the membrane and made available for Wg signaling (Greaves, 1999).

The fat gene negatively controls cell proliferation in a cell autonomous manner. The Fat protein (with 5,147 amino acids) contains four major regions. Beginning by the N-terminus there are 34 cadherin-like domains, five EGF-like repeats interspersed with two laminin A-G chain motifs, a transmembrane domain and a novel cytoplasmic domain (Mahoney, 1991). Several cell behavior parameters of mutant alleles of fat ( ft) have been studied in Drosophila imaginal wing disc development. Mutant imaginal discs continue growing in larvae delayed in pupariation and can reach sizes of several times those of wild-type. Their growth is, however, basically allometric. Homozygous ft cells grow faster than their twin cells in clones and generate larger territories, albeit delimited by normal clonal restrictions. Moreover, ft cells in clones tend to grow towards the wing proximal regions. These behaviors can be related with failures in cell adhesiveness and cell recognition (Garoia, 2000).

The cadherins Fat and Dachsous regulate dorsal/ventral signaling in the Drosophila eye

Planar polarity refers to the asymmetry of a cell within the plane of the epithelium; for example, cells may form hairs that point in a posterior direction, or cilia may beat uniformly. This property implies that cells have information about their orientation; it is of interested to discover the nature of this information. Relevant also is the body plan of insects, which, in the ectoderm and somatic mesoderm, consists of a chain of alternating anterior and posterior compartments -- basic units of development with independent cell lineage and subject to independent genetic control. Using the abdomen of adult Drosophila, genes required for normal polarity were either removed or constitutively expressed in small clones of cells and the effects on polarity were observed. Hitherto, all such studies of polarity genes have not found any difference in behavior between the different compartments. This study shows that the three genes, four-jointed, dachsous, and fat, cause opposite effects in anterior and posterior compartments. For example, in anterior compartments, clones ectopically expressing four-jointed reverse the polarity of cells in front of the clone, while, in posterior compartments, they reverse behind the clone. These three genes have been reported by others to be functionally linked. This discovery impacts on models of how cells read polarity. At the heart of one class of models is the hypothesis that cell polarity is determined by the vector of a morphogen gradient. Evidence is presented that cell polarity in the abdomen depends on at least two protein gradients (Fj and Ds), each of which is reflected at compartment borders. Consequently, these gradients have opposing slopes in the two compartments. Because all polarized structures made by abdominal cells point posteriorly, it is surmised that cells in each compartment are programmed to interpret these protein gradients with opposite signs, pointing up the gradient in one compartment and down the gradient in the other (Casal, 2002).

Two other genes resemble fj with regard to compartment-specific effects: dachsous (ds) and fat (ft). In both cases, UAS transgenes cannot be easily made, so only the effects of removing the genes have been studied. Dachsous is a giant integral membrane protein with many cadherin domains. ds gene expression has been monitored using a ds.lacZ transgene. In each segment of the tergites, ds.lacZ is expressed in one band per metamere with a peak near the A/P border that extends into both compartments. This single band is more clearly apparent in the pleura and appears to be centered in a more anterior location than in the tergite or sternite (Casal, 2002).

ds^- flies are lethal, but some hypomorphic mutants survive to adulthood with defective limbs -- the tarsi show polarity defects. In the abdomen of these flies, the anterior parts (a2) of the A compartments are fairly normal, but much of the rest of the A and P compartments is affected by whorls. Remarkably, hair orientation in the back half of the P compartments, both dorsal and ventral, is reversed (Casal, 2002).

In the tergites, ds^- clones are characterized by whorling hairs within the clone. They cause some swirly repolarization of the hairs in front of the clone in the A compartment, but not behind. These whorls could indicate that there has been a loss of overall polarity, even though some local coordination between adjacent cells remains. In the P compartment, ds^- clones induce clear reversal of hairs behind the clone without affecting the front. Just as with clones ectopically expressing fj, those situated at the back boundary of the A compartment reorient hairs outside the clone, both anterior to the clone (A cells) and posterior to it (P cells) — but hairs within the clone are more whorly than with fj-expressing clones (Casal, 2002).

Thus, apart from the whorls, ds^- clones are reminiscent of UAS.fj clones; both cause non-autonomous reversals in opposite ways in the A and the P compartment. Accordingly, Ds, like Fj, may form opposing gradients in A and P, each being interpreted with opposite signs. Because loss of Ds activity mimics gain of Fj activity, it is deduced that the gradients of Fj and Ds activity are reciprocal to each other, a conclusion that fits with the expression pattern of both genes in the pleura (Casal, 2002).

Like ds, ft encodes a huge molecule with many cadherin repeats, and as with ds, null mutant flies do not develop. The mutant imaginal discs grow excessively, and there are some effects on the polarity of bristles. Clones of ft^- cells in otherwise wild-type discs are abnormally large; in the abdomen, these clones tend to be creased, as if they were trying to grow beyond their normal compass (Casal, 2002).

In the A compartments of the tergites, ft^- clones tend to disturb and reverse polarity behind the clone, while, in the P compartments, they tend to reverse in front. Thus, ft^- clones, like ds^- and fj^- clones, have opposite effects on polarity in A and P. When the ft^- clones are near the A/P boundary, they behave as would be expected from the provenance of the cells neighboring the clone: clones at the back of the A compartment fail to reverse the P cells behind (P cells normally reverse in front of a ft^- clone), and P clones fail to reverse A cells in front of them (A cells normally reverse behind a ft^- clone) (Casal, 2002).

Thus, ft^- clones, like ds^- and fj^- clones, have opposite effects on polarity in A and P. Further, the effects of ft^- clones are similar to those of fj^- clones but are opposite those of UAS-fj and ds^- clones. For example, in the A compartment, hairs point toward ft^- clones but away from UAS-fj clones, whereas, in P, they point away from ft^- clones but toward UAS-fj clones. Using the logic deployed with fj and ds, it is inferred that Ft activity is reflected like that of Fj, forming a peak at the segment boundary and declining to a trough at the A/P boundary. But note that ft^- clones can cause polarity reversals anywhere within A, as well as in anterior P -- but fj^- clones do so only in anterior A. This difference argues for a model in which Fj is produced only by cells flanking the segment boundary, acting non-autonomously on cells further away, whereas Ft activity might be required autonomously in all cells, with any differential in Ft activity between neighboring cells determining their polarity (Casal, 2002).

The three genes ds, ft, and fj are functionally linked: mutations in all three damage the tarsi in a similar way; ds and ft encode similar cadherin molecules, and they and fj interact genetically. For the Drosophila eye, it has been proposed that the products of ds, ft, and fj work together in a linear pathway in the developing ommatidia. This pathway begins with a gradient of Wg and leads to the differential activation of Fz in the presumptive R3 and R4 cells. According to this model, graded Wg spreads into the eye from sources at the dorsal and ventral poles, induces Ds expression, represses Fj expression, and thereby generates reciprocal Ds and Fj gradients. Fj activity then represses Ds activity and reinforces this reciprocity. In turn, the Ds gradient then patterns the activity of Ft, which is ubiquitously expressed. Finally, the gradient of Ft activity promotes the activation of Fz in the more equatorial cell and directs it to become the R3 cell, while the more polar cell becomes the R4 cell (Casal, 2002).

The present results point to parallels between the action of Fj, Ds, and Ft in the eye and abdomen. In both cases, a morphogen (Wg in the eye, and Hh in the abdomen) appears to govern polarity through the induction of reciprocal gradients of Fj and Ds expression. Further, in the abdomen, Hh organizes polarity at least in part through the induction of Wg. Hence, as in the eye, peak Wg activity occurs where fj is repressed and where ds is expressed. Finally, the results suggest that the gradient of Ds in the abdomen is reciprocal to that of Ft activity, consistent with the model proposed for the eye. These parallels suggest that the three genes are part of a mechanism common to the eye and abdomen and presumably elsewhere (Casal, 2002).

These results argue that, in the abdomen, the compartmental provenance of responding cells is crucial. This is particularly clear for clones that either lack or overexpress fj. It is found that, in the A compartment, hairs point down gradients of Fj activity, while, in the P compartment, they point up. This discovery can help explain how all cells in the abdominal epidermis make hairs that have the same polarity, even though, in both compartments, the gradients of Fj and Ds decline in opposite directions. However, it presents other problems (Casal, 2002).

One problem is that it has been proposed that Hh drives polarity by inducing a gradient morphogen, X, whose slope specifies polarity. The model is that Hh enters the A compartment from the P compartment behind it and acts through wg and optomotor blind (omb) to induce X. For simplicity, it was conjectured that X might form a monotonic gradient, spreading forward from its peak at the back of the A compartment all the way to the front of the P compartment of the next segment. According to this conjecture, all cells in both A and P make structures that point posteriorly because all respond to the common vector of a monotonic gradient of X (Casal, 2002).

However, the present results argue for reflected gradients centered around the A/P compartment boundary and against a monotonic gradient for X. Thus, it is now surmised that Hh induces reflected gradients of Fj, Ds, and Ft activity. It is instructive to compare the imaginal discs with the abdomen. In the discs, unidirectional Hh signaling across the A/P boundary induces the morphogens Decapentaplegic (Dpp) and Wg, and these then spread both anteriorly and posteriorly and create reflected gradients that pattern both compartments. In the abdomen, Hh also induces Wg (in the tergites and sternites) and Dpp (in the pleura). At least in the tergites, Wg then spreads posteriorly from its source at the back of the A compartment to induce omb and specify cell pattern in the P compartment. Thus, the combined activities of Hh in A cells, and of Hh-induced Wg moving back into P cells, generates a zone of Omb expression spanning the A/P boundary. It is now suggested that this band of Omb organizes the reflected gradients of Ds and Fj, which in turn, through Ft, help polarize the cells. Thus, the combined actions of Fj, Ds, and Ft might constitute what was previously called X (Casal, 2002).

Another problem is raised by the finding that cells in the A and P compartments interpret the polarizing activities of Fj, Ds, and Ft with opposite response. In the wing, gene products such as Fz and Dsh accumulate transiently along the distal edge of each cell and forecast both the site and distal direction of hair outgrowth. Further, wing hairs invariably point away from UAS.fz clones and toward fz^- clones, and this suggests that these subcellular localizations could be a readout of differential Fz activity. It was found that UAS.fz and fz^- clones in the abdomen behave like their counterparts in the wing, whether in the A or P compartment -- in all cases, hairs point away from UAS.fz clones and toward fz^- clones. Thus, it is inferred that in the normal abdomen, Fz and Dsh accumulate along the posterior edge of both A and P cells, even though the controlling gradients of Fj, Ds, and Ft in the A compartment have the opposite slopes of those in the P compartment (Casal, 2002).

How might A and P cells be programmed so that bidirectional activity gradients of Fj, Ds, or Ft would lead to a unidirectional slope of Fz activity? It is suggested that a transcription factor, Engrailed, encoded by the selector gene that distinguishes P from A cells, also alters the response of P cells relative to A cells, so that in A cells, Fz might accumulate at the cell edge where Fj is lowest, while, in the P cells, it might accumulate where it is highest. The result would be a localized accumulation of Fz along the posterior edge in all cells, whether in A or P. A precedent comes from yeast, where haploid (a or a) cells bud axially near prior budding sites, while diploid (a/a) cells bud in a bipolar fashion at the site farthest from the previous bud. In yeast, this switch in polarity is also governed by transcription factors encoded by the mating-type locus (Casal, 2002).

In the abdomen, there are observations that do not fit with a simple linear pathway as proposed for the eye. For example, hair polarities are not randomized in fj^-, ds^-, or ft^- mutant tissues, and even entirely fz^- flies show relatively normal polarity in most regions. Nevertheless, consistent changes in polarity are generated by disparities in the activity of each of these polarity genes, usually across clone borders. Hence, cell polarity may depend on multiple signals of which the mutually reinforcing effects of Fj and Ds are but one example (Casal, 2002).

An axon scaffold induced by retinal axons directs glia to destinations in the Drosophila optic lobe

In the developing Drosophila visual system, glia migrate into stereotyped positions within the photoreceptor axon target fields and provide positional information for photoreceptor axon guidance. Conversely, glial migration depends on photoreceptor axons, as glia precursors stall in their progenitor zones when retinal innervation is eliminated. These results support the view that this requirement for retinal innervation reflects a role of photoreceptor axons in the establishment of an axonal scaffold that guides glial cell migration. Optic lobe cortical axons extend from dorsal and ventral positions toward incoming photoreceptor axons and establish at least four separate pathways that direct glia to proper destinations in the optic lobe neuropiles. Photoreceptor axons induce the outgrowth of these scaffold axons. Most glia do not migrate when the scaffold axons are missing. Moreover, glia follow the aberrant pathways of scaffold axons that project aberrantly, as occurs in the mutant dachsous. The local absence of glia is accompanied by extensive apoptosis of optic lobe cortical neurons. These observations reveal a mechanism for coordinating photoreceptor axon arrival in the brain with the distribution of glia to multiple target destinations, where they are required for axon guidance and neuronal survival (Dearborn, 2004).

Attempts were made to determine the location of progenitors that give rise to the distinct types of migratory glia and the neurons that form their migratory pathways. The Wingless expressing cells of the dorsal and ventral domains are located in areas of complex gene expression controlled by Wingless (Wg) signaling activity. Adjacent to the Wg domains are non-overlapping cell populations that express the TGF-ß family member Decapentaplegic (Dpp). Both the Wg- and Dpp-positive cell populations express the transcription factor Optomotor Blind (Omb). Dachsous (Ds), a Cadherin family member, is expressed in a graded fashion with respect to the Wg domains. These three genes, though expressed in different patterns, are under the control of Wg activity (Dearborn, 2004 and references therein).

It is concluded that Drosophila optic lobe glia use axon fascicles as migratory guides and that the extension of these axon fascicles is induced by the ingrowth of photoreceptor axons from the developing retina. The migratory scaffold axons emerge from optic lobe regions that are in close proximity to sites where glial cells originate; both arise in the dorsal and ventral domains where cells express the morphogen Wingless. When the scaffold axons were eliminated by the autonomous expression of an activated Ras transgene, glia failed to migrate and stalled at the borders of their progenitor sites. Extensive cortical cell apoptosis ensued. When the scaffold axons projected aberrantly (in animals mutant for the cadherin Dachsous), glia followed the aberrant routes to incorrect destinations. The longstanding observation that glial migration does not occur in eyeless mutant Drosophila might thus be explained by an indirect mechanism in which innervation controls the establishment of an axon scaffold necessary to direct glial migration (Dearborn, 2004).

Dachsous and planar polarity in the eye

Fat also plays an important role in planar polarity. This phenomenon is evidenced by the coordinated orientation of ommatidia in the Drosophila eye. Planar polarity requires that the R3 photoreceptor precursor of each ommatidium has a higher level of Frizzled signaling than its neighboring R4 precursor. Two cadherin superfamily members, Fat and Dachsous, and the transmembrane/secreted protein Four-jointed play important roles in this process. The data support a model in which the bias of Frizzled signaling between the R3/R4 precursors results from higher Fat function in the precursor cell closer to the equator -- the cell that becomes R3. Evidence is also provided that positional information regulating Fat action is provided by graded expression of Dachsous across the eye and the action of Four-jointed, which is expressed in an opposing expression gradient and appears to modulate Dachsous function. It is suggested that the presence of relatively higher Ds function in the polar cell could result in a difference in Ft function between the R3/R4 precursors by either inhibiting Ft function in a cell-autonomous fashion or by stimulating Ft function in the equatorial cell. The difference in Ft function between the precursor cells biases Fz signaling so that the equatorial cell has higher Fz activity (Yang, 2002).

The lack of a graded Ft expression suggested that Ft might be regulated by proteins that are themselves expressed in gradients in the eye disc. Two previous findings prompted a test of the role of dachsous (ds, which encodes a Fat-like transmembrane protein containing 27 cadherin repeats in its extracellular domain), as a Ft regulator: (1) loss of Ds function in the wing causes planar polarity defects; (2) removal of a single copy of ds suppresses defects caused by a dominant ft mutation, suggesting that Ds might act in conjunction with Ft. To determine whether Ds provides positional information regulating Ft function in the eye, ds expression was examined in the eye imaginal disc using a ds-lacZ enhancer trap (a ß-gal reporter gene inserted in the first intron of ds) that faithfully reproduces ds expression during third instar larval development. Interestingly, ds-lacZ is expressed in a graded pattern that is high at the two poles and low at the equator. In order to confirm that this gradient of ds transcription results in a gradient of Ds protein expression, antibodies were raised against the intracellular domain of Ds and used to stain eye discs. In the region near the morphogenetic furrow where the R3 and R4 fates are being specified, graded expression of Ds from the poles was readily apparent. In older ommatidia in the posterior region of the disc, Ds, like Ft, accumulates in a subset of cells surrounding each ommatidium (Yang, 2002).

If Ds is an important regulator of Ft, then the absence of Ds should lead to randomized d-v polarity. This prediction was examined using animals homozygous for the ds loss-of-function mutation ds^UA071. While Ds function is important for normal viability, a few homozygous ds^UA071 mutant animals survive for a few days after eclosion. Similar to ft ommatidia, the ds ommatidia randomly adopted either d or v polarity. Similar results were observed in animals transheterozygous for ds^UA071 and another strong ds allele (ds^38K) and in marked clones of homozygous ds^UA071 cells (~40% polarity reversals). The examination of ds mutant clones also showed the presence of nonautonomous effects on the polarity of neighboring wild-type tissue along the equatorial border of the clone (Yang, 2002).

When the pattern of R3/R4 specification and Fz signaling in ds eye discs was examined by staining for either the E(spl)mdelta0.5 marker or Fmi, the results were very similar to those described above for ft mutant clones. In both cases, the mutant ommatidia exhibited polarized Fmi localization and R4 reporter expression, but the direction of polarization was randomized. Thus, Ds, like Ft, plays an essential role in the establishment of properly biased Fz signaling during R3/R4 specification (Yang, 2002).

The requirement for Ds during the establishment of ommatidial polarity and the gradient of ds expression suggested that higher ds expression in the polar member of the R3/R4 precursor pair might contribute to the normal pattern of R3/R4 specification by modulating Ft function. For example, the presence of higher Ds in the polar precursor cell might either inhibit Ft function within the polar cell or else promote Ft function in the neighboring equatorial precursor cell. This proposal makes several predictions: (1) loss of Ds function from the polar cell, which would reverse the relative levels of Ds within an R3/R4 precursor pair, should lead to reversals in R3/R4 specification pattern and reversals of ommatidial polarity; (2) in contrast, loss of Ds from the equatorial cell, which does not change the direction of the ds gradient within an ommatidium, should have little effect on polarity; (3) loss of Ds from the polar cell should have no effect if that cell also lacks functional Ft (Yang, 2002).

To analyze the effects of a lack of Ds function in one member of an R3/R4 precursor pair, homozygous ds^UA071 clones were generated in heterozygous animals and R3/R4 mosaic ommatidia were examined. The loss of Ds function from the equatorial R3/R4 precursor cell had only a mild effect on ommatidial polarity (13% polarity reversals in 127 examples. In contrast, loss of Ds function from the polar cell led to polarity reversals in 43% of 98 examples. These data demonstrate that Ds acts primarily in the polar precursor cell and are consistent with the idea that the graded ds expression contributes to the normal pattern of R3/R4 specification by providing a higher Ds level to the polar cell of each R3/R4 precursor pair. However, the observation that differences in Ds function between the R3/R4 precursor cells are less effective than Ft differences at directing the pattern of R3/R4 specification suggests that, in addition to its primary role in the polar precursor cell, Ds may also play a role in regulating Fz signaling from the equatorial cell (Yang, 2002).

In order to test whether Ds regulates Fz signaling by modulating Ft function, the genetic interaction of ft and ds in specifying R3/R4 cell fates was examined. Since removal of Ds from the polar precursor cell frequently causes this cell to become R3 rather than R4 while the absence of Ft from an R3/R4 precursor cell leads to its specification as R4, the effects of removing both Ds and Ft from the polar cell were examined using marked clones of ds^UA071;ft^Gr-V double mutant cells. Polarity reversals occurred in only 2.5% (2 out of 80 examples) of the R3/R4 mosaic ommatidia in which the polar precursor cell lacked Ds and Ft. This result contrasts with the 43% polarity reversals observed when only Ds function was lost from the polar cell. Indeed, the effect of removing both Ds and Ft functions was remarkably similar to that of removing Ft alone. In each case, the mutant precursor cell was preferentially specified as R4 (80% and 98%, respectively). Thus, the absence of Ft function from one R3/R4 precursor cell determines the pattern of R3/R4 specification in a manner that is largely independent of input from Ds. These results suggest that Ds acts upstream of Ft and are consistent with the idea that the presence of higher Ds levels in the polar R3/R4 precursor directs correct ommatidial polarity by ensuring that Ft activity is higher in the equatorial precursor cell (Yang, 2002).

To further explore how Ds might act in regulating Ft, it was asked whether Ft protein level is altered in ds mutant clones. Staining for Ft was carried out in ds clones and it was found that the level of Ft is increased slightly within the clones. This result suggests that one mode of Ds regulation might be to antagonize Ft protein expression or stability (Yang, 2002).

This analysis supports the idea that positional information controlling Fz signaling during ommatidial development is provided by the opposing gradients of fj and ds expression. The question arises as to how these gradients are established. Previous work has shown that a major determinant of the fj expression gradient is Wg, a secreted Wnt class ligand that negatively regulates fj expression and that is expressed at high levels at the two poles of the eye disc. To test whether the Wg gradient also contributes to the regulation of ds expression, clones of cells in which Wg signaling was either ectopically activated or reduced were examined in animals carrying the ds-lacZ reporter. Ectopic activation was achieved by overexpressing a constitutively activated form of Armadillo (Arm) and resulted in a dramatic increase in ds-lacZ expression. The effects of attenuating Wg signaling were assayed in clones of cells homozygous for the hypomorphic arm^H8.6 mutation. ds-lacZ expression was severely reduced in these clones. Combined with previous studies of fj-lacZ expression, these data suggest that the ds and fj expression gradients result in large part from the presence of a gradient of Wg signaling that increasingly activates ds and inhibits fj expression near the poles. It is worth emphasizing that the receptor mediating the effects of Wg on fj and ds expression is likely to be another member of the Fz family, perhaps dFrizzled2 (dFz2), rather than Fz itself. This is evident from the observation that fj-lacZ expression is not affected by the loss of Fz function (Yang, 2002).

Another study suggests that fat is involved not in the establishment of R3 and R4 cell fate, but instead functions in establishing the dorsal-ventral midline (equator) during eye morphogenesis. The Drosophila eye is a polarized epithelium in which ommatidia of opposing chirality fall on opposite sides of the eye's midline, the equator. The equator is established in at least two steps: photoreceptors R3 and R4 adopt their fates, and then ommatidia rotate clockwise or counterclockwise in accordance with the identity of these photoreceptors. Two cadherins, Fat (Ft) and Dachsous (Ds), play a role in conveying the polarizing signal from the D/V midline in the Drosophila eye. In eyes lacking Ft, the midline is abolished. In ft and ds mutant clones, wild-type tissue rescues genetically mutant tissue at the clonal borders, giving rise to ectopic equators. These ectopic equators distort a mosaic analysis of these genes and has led to a possible misinterpretation that ft and ds are required to specify the R3 and R4 cell fates, respectively (Yang, 2002). Interpretation of these data supports a significantly different model in which ft and ds are not necessarily required for fate determination. Rather, they are involved in long-range signaling during the formation of the equator, as defined by the presence of an organized arrangement of dorsal and ventral chiral ommatidial forms (Rawls, 2002).

ft has long been known for its role in proliferation control. The identification of new ft alleles in a FLP/FRT screen has revealed a role for Ft in establishing epithelial polarity. In ft⁴²² null clones, approximately 52.5% of ommatidia—including mosaic and genetically mutant ommatidia—exhibit defects in polarity. Of these, 50.5% are inverted on their D/V axis. The remaining 2.0% of ommatidia are inverted on their A/P axis or on both their A/P and D/V axes. Furthermore, 98% of mosaic ommatidia that are phenotypically mutant are inverted on their D/V axis (Rawls, 2002).

Dorsoventrally inverted ommatidia are not randomly distributed within ft clones. Rather, they are preferentially localized toward the polar border such that the phenotypically mutant ommatidia are found in the polar region of the clone and phenotypically wild-type ommatidia are found along the equatorial border. The consequence of this biased distribution of ommatidia is an 'inverted equator' (originally called a pseudoequator) within the mutant clone, in which the points of opposing trapezoids face each other. Inverted equators in ft clones consistently arise approximately two rows from the equatorial border of the clone. These inverted equators were seen in 35/41 (85%) ft⁴²² clones. The 15% of ft clones with no apparent ectopic equator were either small or long and narrow and therefore not broad enough to detect this phenotype (Rawls, 2002).

While a small percentage of ft clones lie along the equator, of over 200 ft clones examined, none cross the equator. A closer examination of these clones has revealed that the position of the endogenous equator, which can be identified in neighboring wild-type tissue, gets shifted by one to two ommatidial rows along the mutant border to accommodate the ft mutant clone. These observations suggest that the juxtaposition of ommatidia with high versus low Ft activity influences the placement of the equator (Rawls, 2002).

ds, known for its role in morphogenesis, plays a role in setting up polarity in the eye. The bias of D/V:A/P errors is similar to that described for ft clones, although fewer ommatidia are disrupted. Approximately 29% of ommatidia (both mosaic and genetically mutant) in ds^38K clones (strong hypomorphic allele) display D/V inversions, while fewer than 1% of genetically mutant ommatidia display A/P inversions (517 ommatidia scored from 12 ds^38k clones). Inverted equators also occur in ds mutant clones at a similar frequency as is seen with ft: 15 out of 18 clones (83%) had inverted equators. However, it is interesting to note that ds inverted equators arise along the equatorial border of the clone rather than within the clone, as is seen with ft. Finally, although ectopic ds equators are rare, they do occur. This phenotype might be more penetrant in a null allele of Ds function; however, no such alleles have been reported (Rawls, 2002).

Inverted and/or ectopic equators have also been observed in mirr, fng, and four-jointed (fj). One significant difference between ft/ds and these genes is that, in these other examples, mutant tissue nonautonomously disrupts wild-type tissue, generating a contiguous patch of nonautonomous D/V inversions. In ft, nonautonomous effects are never observed in wild-type ommatidia. In ds, the nonautonomy can extend many rows beyond the clone and may be separated by up to eight rows of unaffected ommatidia. It is appealing to speculate that this 'extended' nonautonomy is an effect of the twin-spot clone, in that a difference in relative amounts of Ds activity reverses the polarity. However, the scattered occurrence of these inverted ommatidia and the fact that a range cannot be seen in the number of affected ommatidia make this hypothesis somewhat unsatisfying. In order to test this 'twin-spot' hypothesis unambiguously, again, a protein null allele of ds is necessary (Rawls, 2002).

The clonal phenotypes of ft and ds suggest that they are involved in establishing equators. Since the establishment of the equator is known to involve long-range signaling, the nonautonomous effects seen in the clonal phenotypes might be a consequence of a requirement for ft and ds in the transduction of a global patterning signal. To remove any effects of long-range signaling, mutant eyes were generated completely devoid of wild-type Ft using the EGUF system (Stowers, 1999). In contrast to ft clones, in EGUF-ft eyes, the D/V axis is so severely perturbed that the endogenous equator is abolished. The greater degree of disruption observed in EGUF-ft eyes compared to mosaic clones suggests that wild-type tissue communicates with mutant tissue, perhaps via cell-cell relay of the signal transduced by Ft. If this is the case, then the presence of the inverted equator within the mutant clones is established as a consequence of signaling from wild-type tissue (Rawls, 2002).

The EGUF-ds phenotype mimics the clonal ds phenotype: 31% of ommatidia display D/V inversions. In EGUF-ds eyes, the endogenous equator is evident. This may be due to the weaker nature of this allele. Alternatively, it may suggest that Ds plays a more modulating role in establishing the D/V midline than does Ft (Rawls, 2002).

Ft and Ds act nonautonomously in the eye. In ft clones, the majority of polarity defects occur in the polar region of the clone. In contrast, ommatidia in the equatorial region of the clone are phenotypically wild-type, suggesting that wild-type ommatidia outside the equatorial boundary of the clone rescue genetically mutant ommatidia within the equatorial region of the clone. Furthermore, wild-type tissue on the polar border of the clone does not rescue mutant ommatidia within the polar region of the clone, indicating rescue takes place only in an equatorial to polar direction and not from the poles to the equator. If this is the case, the Ft signal is propagated in a directional fashion from wild-type tissue at the equatorial border into the mutant clone. Finally, mutant tissue never nonautonomously affects wild-type tissue -- in over 200 clones analyzed, no inverted ommatidia were seen in which all eight photoreceptors were wild-type. In contrast to ft, rescue takes place in a polar to equatorial direction in ds clones (Rawls, 2002).

The tissue polarity genes fz and stbm are required to specify R3 and R4. These cells then regulate the direction of ommatidial rotation. Given the importance of these two cells in the establishment of polarity, ommatidia were examined that were mosaic for ft within the R3 and R4 pair. In the majority of cases, the Ft⁺ cell becomes R3. A similar analysis of the other developmental pairs of photoreceptors, R1/R6 and R2/R5, revealed that there is a strong tendency for the Ft⁺ cell to adopt the fate of the anterior (R1 and R2) rather than the posterior (R5 and R6) photoreceptor cell (Rawls, 2002).

There are two possible interpretations of these data. First, ft may be involved in specifying the anterior photoreceptor fates, as the data imply. However, if Ft is required to specify the R3 fate, at least a small fraction of ommatidia should be seen in which no R3 fate is specified in ft mutant tissue (i.e., ommatidia that have two R4s), as is the case in fz mutants, this phenotype is not seen in ft clones. Alternatively, this finding could reflect the link between how cells are recruited into the growing ommatidium, how ommatidia rotate to establish polarity, and how this process is disrupted in ft mutant ommatidia (Rawls, 2002).

In doing this mosaic analysis, it was essential to recognize that a property inherent to eye development is that ommatidia that arise at the polar border of a clone predominantly recruit their polar cells from wild-type tissue and their equatorial cells from mutant tissue. Phenotypically mutant ommatidia occur only in the polar region of ft clones. This phenotype complicates the analysis and makes it difficult to draw conclusions regarding the specific cell(s) in which Ft is required for cell fate (Rawls, 2002).

In wild-type clones, the cells that are recruited from the polar side of the clone (R4, R5, and R6) will face the posterior side of the clone at the end of rotation. In ft clones, ommatidia that are both phenotypically mutant and mosaic occur only at the polar boundary of the clone. Since these ommatidia are almost always D/V inversions, then they will have recruited their ft⁺ cells from the polar side of the clone, but rather than these polar-derived cells becoming posterior photoreceptors (R4, R5, and R6) as they would have in wild-type, they become anterior photoreceptors (R1, R2, and R3). Together, these factors create an artifactual bias in which the anterior photoreceptor cells are ft⁺ and the posterior cells are ft^-. Consequently, even though the data appear to indicate ft is required for the anterior cell fates (R1, R2, R3), a more detailed analysis reveals that the nature of the ft phenotype introduces a developmental bias that must be considered (Rawls, 2002).

The mosaic analysis of ds mutant clones revealed a trend the opposite of that described for ft. In ommatidia mosaic for ds in the pairs R1/R6, R2/R5, and R3/R4, the majority of photoreceptors are wild-type for the posterior fates (R4, R5, and R6). Since D/V inversions are found on the opposite side of the clone in ds compared to ft, this is the expected result if one applies the same logic as described above for ft. As with ft, no functional autonomy can be assigned to a single cell (Rawls, 2002).

A contrasting interpretation of a mosaic analysis of ft and ds, presented by Yang (2002), suggests ft and ds are required to specify the fates of photoreceptors R3 and R4, respectively. However, mosaic analyses of ft and ds are inherently biased due to the clonal phenotypes, as described above. This bias might mask a role for ft or ds in the R3/R4 fate decision, but currently there is no compelling evidence for such a functional requirement. The genetic data reported here and in Yang (2002) are insufficient to draw conclusions about the role of ft and ds in fate specification. Extensive experimentation and a better understanding of mechanism are necessary to discriminate between a role for ft in global D/V signaling versus a requirement for ft and ds in specification of the R3 and R4 cell fates (Rawls, 2002).

Early acting genes, for example, mirror (mirr) and fringe (fng), specify the dorsal and ventral halves of the eye, respectively, thereby setting up the D/V boundary. The ft clonal phenotype suggests ft might mediate D/V boundary formation. To address this possibility, the effect of ft on mirr expression was assessed. The expression pattern of the enhancer trap line 8A5, in which the mirr promoter drives expression of white, is unaffected in EGUF-ft eyes. In these eyes, mirr expression remains restricted to the dorsal half of the eye, indicating that ft is required either downstream of or in parallel to mirr (Rawls, 2002).

A proposed model for ft activity differs from that of Yang (2002) because this study (1) takes into account the observations that ectopic equators are generated in mutant clones and that the ft clonal phenotype differs significantly from the EGUF-ft phenotype and (2) considers fundamental properties of eye development in conjunction with the clonal phenotype. A model is proposed in which ft conveys D/V positional information to developing ommatidia to create the D/V midline (Rawls, 2002).

Ft functions to inhibit D/V signaling in the wing and haltere. The data are consistent with this proposal -- new equators are generated in ft mutant clones in the eye. It is proposed that a consistent level of Ft activity throughout the eye inhibits the D/V signaling required to form the equator. At the equator, Ft activity must be inhibited. The molecule that inhibits Ft could be expressed in several ommatidial rows encompassing the future midline. In a ft mutant clone, the phenotype is rescued for two rows in the equatorial region of the clone, suggesting the D/V signal propagated by Ft can be relayed for a distance of two ommatidial rows. Therefore, it is proposed that in the presence of the regulatory protein, the D/V signal can also be relayed an equivalent distance at the endogenous equator. When the D/V signal reaches its minimum, tissue with Ft activity apposes tissue without Ft activity, and it is at this point that the equator is established (Rawls, 2002).

Nonautonomous planar polarity patterning in Drosophila: Dishevelled-independent functions of Frizzled

The frizzled (fz) gene of Drosophila is required for planar polarity establishment in the adult cuticle, acting both cell autonomously and nonautonomously. These two activities of fz in planar polarity are temporally separable in both the eye and wing. The nonautonomous function is dishevelled (dsh) independent, and its loss results in polarity phenotypes that resemble those seen for mutations in dachsous (ds). Genetic interactions and epistasis analysis suggest that fz, ds, and fat (ft) act together in the long-range propagation of polarity signals in the eye and wing. Evidence has been found that polarity information may be propagated by modulation of the binding affinities of the cadherins encoded by the ds and ft loci (Strutt, 2002).

In the wild-type wing, each cell produces a single trichome at its distal vertex that points distally. Flies that lack fz function exhibit defects in trichome polarity. Most cells still produce a single trichome, but these are arranged in a distinctive swirling pattern known as the fz/in-like pattern. Furthermore, in the pupal wing, there is no asymmetric localization of the polarity proteins Fz, Dsh, Fmi, Pk, and Dgo and the majority of trichomes form in the cell center. An almost identical phenotype is caused by mutations that remove only the cell-autonomous functions of fz or mutations in the cell autonomously-acting polarity genes dsh and fmi. This indicates that this phenotype is likely to be solely the result of removing cell-autonomous polarity gene function (Strutt, 2002).

In contrast, ds is thought to be required only for the nonautonomous transmission of polarity information. Wings from ds individuals show a trichome swirling pattern, which is distinct from the fz/in pattern. Asymmetric polarity protein localization still occurs—albeit often in an aberrant pattern Similarly, cells adjacent to a fz clone, which have aberrant polarity because of the nonautonomous phenotype of fz, also asymmetrically localize polarity proteins (Strutt, 2002).

On the basis of these observations, it seems likely that the loss of fz function from the entire wing results in both cell-autonomous and -nonautonomous phenotypes, but that the former masks the latter. To study the phenotype caused by loss of the nonautonomous component only, a method of rescuing only the cell-autonomous phenotype must be devised. It was hypothesized that the nonautonomous function of fz might precede the cell-autonomous function. If this is correct, it should be possible to rescue only the cell-autonomous function of fz by adding back fz activity to a fz mutant fly at a stage of development after the nonautonomous requirement, but before the autonomous requirement. To test this, a transgene was used consisting of the Actin5C promoter separated from the fz coding sequence by an FRT-flanked transcription termination sequence. The fz coding sequence is fused to the coding sequence of GFP, to permit monitoring of fz expression, giving rise to the transgene Actin>stop>Fz-GFP. Expression of Fz-GFP from this transgene can be induced by heat-shocking flies that also carry a transgene containing yeast FLP recombinase under control of the hsp70 promoter (Strutt, 2002).

This expression system was used to rescue the phenotype of strong fz mutations that lack both cell-autonomous and -nonautonomous activity. Activation of Fz-GFP expression at time points up to about 6 hr after prepupa formation (APF) results in a wild-type trichome polarity pattern. However, activation of Fz-GFP expression at about 6 hr APF results in a weak polarity defect in some parts of the wing. Successively later activation results in an increasingly strong trichome polarity phenotype. Remarkably, this trichome polarity phenotype appears more ds-like than fz/in-like, in particular, showing complete inversions of trichome polarity in some proximal regions of the wing. Activation of Fz-GFP expression at 16 hr APF produces a pattern most similar to that seen in strong ds mutants. Activation at 24 hr APF gives a phenotype that is interpreted as a stronger form of the reported ds-like pattern (and that is stronger than the fz/in pattern). Notably, the characteristic wing shape and vein phenotypes seen in ds mutations are not observed, suggesting that the effects of ds on trichome polarity and wing morphology could be separable functions (Strutt, 2002).

Activation of Fz-GFP expression at later time points results in no increase in strength of the ds-like polarity phenotype. Instead, after 27 hr APF, progressively weaker phenotypes are seen that resemble a mixture of the ds-like and fz/in-like patterns, until about 31 hr APF, when the adult polarity pattern is typically fz/in-like and is identical to that produced in the absence of Fz-GFP expression (Strutt, 2002).

The effects of expression of Fz-GFP at different time points were also examined in the pupal wing at the time of trichome initiation (about 32 hr APF), monitoring Fz-GFP localization and trichome formation. Activation of Fz-GFP expression up to about 24 hr APF results in asymmetric Fz-GFP localization in an aberrant pattern and trichome formation at the corresponding cell edge. Activation at time points from 24 hr APF onward results in reduced asymmetric protein localization and an increase in the number of trichomes initiating away from the cell edge, until, by 30 hr APF, little Fz-GFP expression is seen at the time of trichome initiation, and trichomes are forming at the cell center (Strutt, 2002).

Thus, the temporal requirement for fz shows two phases. Activation of Fz-GFP expression prior to 6 hr APF elicits complete rescue of fz functions. Between 6 and 24 hr APF, lack of fz activity results in a ds-like polarity pattern (which gets progressively stronger, the longer fz activity is not present), but asymmetric polarity protein complexes still form, and trichomes emerge at the corresponding sites at the cell periphery. Between 24 and 31 hr APF, loss of fz activity results in a progressively more dominant fz/in-like trichome polarity pattern, failure to form asymmetric polarity protein complexes, and formation of trichomes in the cell center (Strutt, 2002).

Consistent with the hypothesis that the early phase of fz activity is required for nonautonomous polarity signaling, expression of Fz-GFP at 12 hr APF largely rescues the phenotype of an fz allele that is classified as lacking only cell-autonomous activity. At first sight, it is surprising that such an allele is not completely rescued. However, it has recently been reported that such alleles are also partially deficient in nonautonomous signaling activity (Strutt, 2002).

The results show that there are two temporally separable activities of fz. The later corresponds to the well-characterized fz cell-autonomous function. The early fz activity, the loss of which produces a more ds-like phenotype, is the fz nonautonomous pathway. Notably, this early activity is required over a significant period of time (16-18 hr in the eye and wing). In addition, this nonautonomous activity is dsh independent in the eye (Strutt, 2002).

A number of conclusions follow from these observations: (1) fz exhibits a similar dsh-independent nonautonomous activity in two different tissues (eye and wing), and, therefore, this is likely to be a conserved pathway; (2) the similarity of the fz nonautonomous phenotype to the ds phenotype and genetic interactions between these loci suggests that these molecules might cooperate in a common mechanism to propagate polarity signals; (3) since the nonautonomous activity of fz precedes the autonomous activity, the former is apparently not dependent on the latter. This supports models of polarity patterning, in which a long-range signal is propagated through the tissue prior to the cell-autonomous response to that signal (Strutt, 2002).

It is noted that, although these two activities of fz are temporally separable, during normal development it is likely that the period of nonautonomous activity nevertheless overlaps the beginning of autonomous polarity functions (Strutt, 2002).

In the eye, the clonal phenotypes of long-range patterning factors, such as the canonical Wnt pathway, JAK/STAT, and fj, have led to models in which they act to establish an activity gradient of a polarity signal (the 'secondary signal'). There are a number of reasons for supposing that these three pathways perform distinct functions that differ from that of the fz nonautonomous activity and that fz is likely to act downstream of these other factors: (1) gradients of components of all three of these pathways are apparent in the second instar stage of development, whereas fz nonautonomous activity in the eye is required over a period of up to 16 hr in the third instar; (2) fz nonautonomous activity is epistatic to (functions downstream of) dsh nonautonomous activity, and there are no genetic interactions between fz nonautonomous activity and canonical Wnt, JAK/STAT, or fj activities; (3) all three activities show similar nonautonomous clonal phenotypes with normal ommatidial polarities in the center of clones, but fz exhibits partial randomization of ommatidial polarities inside the clones (Strutt, 2002).

Conversely, there are a number of reasons for thinking that fz nonautonomous activity in the eye is closely related to ds and ft function. The phenotypes of clones lacking early fz function are similar to those of ds clones and ft clones. Furthermore, there are strong genetic interactions between these factors. Finally, an epistasis test between the clonal phenotypes of fz and ds gives an apparently additive (or possibly synergistic) phenotype. These results are consistent with fz acting jointly with ds and ft in the nonautonomous propagation of polarity information. A similar function for ds has been suggested on the basis of studies in the wing, it having been shown that ds nonautonomously affects trichome polarity and that it is likely to be involved in the maintenance or propagation of an fz-dependent nonautonomous polarity signal (Strutt, 2002).

Thus, overall data from both the eye and wing support fj acting upstream of ds and ft, which then act jointly with fz nonautonomous function in the long-range propagation of polarity information. Uncharacterized mechanisms of intercellular signaling then lead to autonomous activation of fz and assembly of asymmetric polarity protein complexes. Note is taken of the contrast with the recent suggestion that ds and ft act directly upstream of the autonomous function of fz (Strutt, 2002).

Other factors or mechanisms must also be involved in nonautonomous propagation of polarity information, in order to explain all of the observations. For instance, complete loss of fj function does not result in a loss of polarity patterning in the wing, indicating that there must be other upstream patterning factors. Furthermore, clones of fj and ft give stronger nonautononomous phenotypes in a central portion of the wing, whereas ds and fz seem to give rather similar phenotypes throughout. This suggests that there are other modulators of pathway activity that have region-specific effects (Strutt, 2002).

Groups of cells lacking fj function tend to round up into tight foci, appearing to have greater affinity for each other than for their fj-expressing neighbors. Furthermore, in mutant cells abutting fj-expressing neighbors, the cadherins Ds and Ft are preferentially found at the cell junctions touching fj⁺ cells. These observations support the notion that one role of fj in wing patterning is to alter the adhesive properties of cells and also of the cadherins Ft and Ds. It is also noteworthy that loss of ft activity results in Ds no longer being tightly localized in the apical junctional zone of cells and that, similarly, loss of ds seems to result in reduction of apical Ft localization (Strutt, 2002).

It is speculated that a gradient of fj activity in the wing might lead to graded Ds/Ft activity and, hence, cell adhesion. Such a gradient of cell adhesion constitutes a possible mechanism for the long-range transmission of polarity information, although direct evidence for this is lacking. It is noteworthy that fj, ft, and ds mutations also all result in truncations of the wing on the proximodistal axis, and it is possible that this phenotype is in some way due to effects on cell adhesion (Strutt, 2002).

Interestingly, the effect of fj clones on Ds/Ft is cell autonomous. It was suggested that, on the basis of its amino acid sequence and in vitro studies, fj encodes a secreted factor and that this property could explain its nonautonomous phenotypes. These results indicate that at least some functions of fj are cell autonomous (Strutt, 2002).

fourjointed interacts genetically with ft and ds in planar polarity and proximodistal patterning

four-jointed (fj) is required for proximodistal growth and planar polarity in Drosophila tissues. It encodes a predicted type II transmembrane protein with putative signal peptidase sites in its transmembrane domain, and its C terminus is secreted. Fj has therefore been proposed to act as a secreted signalling molecule. Fj protein has a graded distribution in eye and wing imaginal discs, and is largely localized to the Golgi in vivo and in transfected cells. Forms of Fj that are constitutively secreted or anchored in the Golgi were assayed for function in vivo. Cleavage and secretion of Fj is shown to not be necessary for activity, and Golgi-anchored Fj has increased activity over wild type. fj has similar phenotypes to those caused by mutations in the cadherin-encoding genes fat (ft) and dachsous (ds). fj is shown to interact genetically with ft and ds in planar polarity and proximodistal patterning. It is proposed that Fj may act in the Golgi to regulate the activity of Ft and Ds (Stutt, 2004).

In Drosophila, the atypical cadherins Ft and Ds are good candidates for being the ultimate targets of fj activity. They are required for both planar polarity and PD patterning, and have similar mutant phenotypes to fj. In addition, fj interacts genetically with ds and ft in both planar polarity and PD patterning. Interestingly, ds fj double mutants have surprisingly strong phenotypes, which were qualitatively different to those of the single mutants, including duplications or transformations of limb structures. However, no such phenotypes are seen in any of the double mutant combinations, suggesting that the duplications/transformations may be specific to the combination of chromosomes used in classical experiments. The current results instead show that mutations in fj enhance the phenotypes of both ft and ds hypomorphic mutations, suggesting that these genes act in a common pathway (Stutt, 2004).

Epistasis experiments further demonstrate that ds is required to mediate fj function, and therefore ds acts downstream of fj; this is in agreement with data based on clonal analysis of ds and fj. Interestingly, recent experiments have also revealed a role for fj in regulating the intracellular distribution of Ds and Ft. In wild-type tissue, Ds and Ft colocalize at apicolateral membranes, and their localization is mutually dependent. Inside fj mutant clones, Ds and Ft localization is largely unaltered. However, in the row of mutant cells immediately adjacent to wild-type tissue, Ft and Ds preferentially accumulate on the boundary between fj⁺/fj^- cells. In addition, cells inside the fj clones appear to be 'rounded-up', suggesting that they prefer to adhere to each other rather than to non-mutant cells. Thus, it is thought that fj modulates the activity and intermolecular binding properties of Ft and Ds (Stutt, 2004).

An interesting point to note is that both ds and ft show planar polarity phenotypes as homozygotes, whereas fj only shows polarity phenotypes on the boundaries of mutant clones. The fj phenotypes have been explained by models in which fj acts redundantly to regulate the production of a gradient, the direction of which determines polarity. Thus, in homozygotes the direction of the gradient is unchanged, and animals show no major defects; but at clone boundaries there is a discontinuity in the direction of the gradient, leading to inversions of polarity. This model can be extended to suppose that Fj may modulate Ds/Ft activity, but that it does not act as a simple on-off switch; rather Ds/Ft retain some activity even when Fj is not present (Stutt, 2004).

In the absence of a known enzymatic function for Fj, the mechanism by which it might modulate Ft and Ds activity remains uncertain. It is speculated that since Fj acts intracellularly, it is possible that it promotes or mediates the post-translational modification of Ds and/or Ft proteins, and that these molecules mediate the non-autonomous signalling functions of Fj. However, the large size of the Ft and Ds gene products (5147 and 3380 amino acids, respectively) renders the analysis of their post-translational modification highly challenging (Stutt, 2004).

Interactions between Fat and Dachsous and the regulation of planar cell polarity in the Drosophila wing

It has been suggested that a proximal to distal gradient of the protocadherin Dachsous (Ds) acts as a cue for planar cell polarity (PCP) in the Drosophila wing, orienting cell-cell interactions by inhibiting the activity of the protocadherin Fat (Ft). This Ft-Ds signaling model is based on mutant loss-of-function phenotypes, leaving open the question of whether Ds is instructive or permissive for PCP. Tools have been developed for misexpressing ds and ft in vitro and in vivo, and these have been used to test aspects of the model. (1) This model predicts that Ds and Ft can bind. Ft and Ds are shown to mediate preferentially heterophilic cell adhesion in vitro, and each stabilizes the other on the cell surface. (2) The model predicts that artificial gradients of Ds are sufficient to reorient PCP in the wing; the data confirms this prediction. (3) Loss-of-function phenotypes suggest that the gradient of ds expression is necessary for correct PCP throughout the wing. Surprisingly, this is not the case. Uniform levels of ds drive normally oriented PCP and, in all but the most proximal regions of the wing, uniform ds rescues the ds mutant PCP phenotype. Nor are distal PCP defects increased by the loss of spatial information from the distally expressed four-jointed (fj) gene, which encodes putative modulator of Ft-Ds signaling. Thus, while the results support the existence of Ft-Ds binding and show that it is sufficient to alter PCP, ds expression is permissive or redundant with other PCP cues in much of the wing (Matakatsu, 2004).

Several gain-of-function findings are consistent with previous loss-of-function findings, and support the model that Ft-Ds signaling is sufficient to influence wing PCP. Ft and Ds preferentially bind in vitro. Patterned misexpression of ds is sufficient to alter wing PCP, consistent with its proposed role as a ligand. The effects of ft or ds misexpression on the direction of hair polarization are usually the opposite of those previously reported from ft or ds loss of function. The direction of hair polarity induced by ectopic ft is usually the opposite of that induced by ectopic ds, consistent with the proposal that Ds binding inhibits Ft activity. Finally, the effects of Ft misexpression are reduced in a ds mutant background, consistent with the proposed role of Ft as a receptor (Matakatsu, 2004),

Nonetheless, the data also show that the proximal to distal gradient of ds expression is not necessary for PCP throughout the wing, despite the distal defects observed in loss-of-function ds mutants. Instead, the experiments show that uniform ds misexpression can rescue the PCP defects caused by a ds mutation in all but the most proximal portions of the wing. Thus, ds is permissive for PCP in most of the wing, and there must be another polarity cue in the distal wing that is sufficient to orient PCP in the presence of uniformly transcribed ds. The experiments indicate that this distal cue is not provided by the distally expressed Fj protein: distal PCP is not disrupted either by uniform misexpression of both ds and fj, or by uniform misexpression of ds in a fj null mutant (Matakatsu, 2004),

It remains possible that the distal cue functions by regulating Ft-Ds signaling. These studies tested the PCP inputs from the patterns of ds and fj transcription, but unknown factors might post-transcriptionally regulate the forms of Ds or Ft protein produced, or their availability at the cell surface. It also is possible that Ft activity is spatially regulated by binding partners other than Ds. ft mutants have stronger PCP and disc overgrowth defects than do ds mutants, and misexpression of ft still causes PCP defects in a ds mutant lacking detectable cell surface protein (Matakatsu, 2004),

Alternatively, the cue may be provided by a mechanism that is completely independent of Ft or Ds. One often-proposed candidate is signaling via the Drosophila Wnts, especially given their patterned (distal or marginal) expression. However, although the misexpression of Drosophila wnt4 can disrupt wing PCP, PCP defects have not been reported in Drosophila Wnt mutants (Matakatsu, 2004),

Although a ds gradient is not required for PCP in most of the wing, it is possible that such a gradient is required locally in the portion of the wing near and proximal to the anterior cross vein. Proximal ds mutant PCP defects could not be rescued with uniform Ds expression, and the data suggest that this is not simply a failure caused by insufficient Ds levels. Thus, the view is favored that this sharp Ds gradient acts as a PCP cue in the proximal wing. If so, this indicates that the cues that orient PCP in the wing are not generally distributed; rather, the wing may be a patchwork of different regions that rely on different cues. This would provide a mechanism for locally altering PCP during evolution without globally affecting polarity in the wing (Matakatsu, 2004),

The hypothesis that Ft acts as a receptor and Ds acts as a ligand for PCP is based, not only on the uniform expression pattern Ft, but also on epistasis experiments in the eye, where the PCP activity of ds clones appears to depend on the presence of ft. Wing PCP can also be disrupted by the expression of a truncated form of Ds lacking its intracellular domain, which is consistent with Ds acting as a ligand (Matakatsu, 2004),

However, this study has shown that misexpressed Ft retains PCP activity in a ds mutant that eliminates detectable cell surface Ds. Thus, Ft activity is apparently n