InteractiveFly: GeneBrief

four-jointed: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

Gene name - four-jointed

Synonyms -

Cytological map position - 55C1--3

Function - a type II transmembrane kinase

Keywords - leg, eye, brain, tissue polarity, phosphorylates of the atypical cadherins Fat and Dachsous, regulates growth via the Hippo/Warts pathway

Symbol - fj

FlyBase ID: FBgn0000658

Genetic map position - 2-81.5

Classification - type 2 transmembrane protein

Cellular location - surface

NCBI link: Entrez Gene
fj orthologs: Biolitmine
Recent literature
Li, C., Li, B., Ma, S., Lu, P. and Chen, K. (2017). Dusky works upstream of Four-jointed and Forked in wing morphogenesis in Tribolium castaneum. Insect Mol Biol. PubMed ID: 28677915
Dusky (dy) is required for cytoskeletal reorganization during wing morphogenesis in Drosophila melanogaster, but which genes participate together with dy for wing morphogenesis has remained unclear. In Tribolium castaneum, dy is highly expressed at the late embryonic stage. Tissue-specific expression analysis indicated high expression levels of dy in the epidermis, head and fat body of late-stage larvae. RNA interference (RNAi) targeting dy significantly decreased adult wing size and caused improper folding of the elytra. Meanwhile, dy knockdown reduced the transcription of four-jointed (fj) and forked (f). These results show that fj RNAi reduces adult wing size and that silencing f results in abnormal wing folding in T. castaneum. Interestingly, knocking down fj and f simultaneously phenocopies dy RNAi, suggesting that dy probably acts upstream of fj and f to regulate wing morphogenesis in T. castaneum.

four-jointed is involved in conveying positional information in the leg and eye, perhaps acting downstream of Notch. fj mutants show reduced growth and altered differentiation only within restricted sectors of the proximal-distal (PD) axis in the leg and wing, thus fj is a candidate for a gene with a coordination function. Consistent with a position-sensitive role, fj is expressed in a regional pattern in the developing leg, wing, eye and optic lobe. The fj gene encodes a novel type II membrane glycoprotein. It is proposed that fj encodes a secreted signal that functions as a positive regulator of regional growth and differentiation along the PD axis of imaginal discs (Villano,1995 and Brodsky, 1996).

Four-jointed was identified in a search for P-element insertions in genes expressed in patterns that might convey positional information in imaginal discs. The beta-galactosidase markers expressed by these P-elements were examined at the late third instar and prepupal stages, a period when axis specification is complete but overt differentiation of each disc has not yet occurred. Based on its provocative expression pattern in the leg disc, eye disc and optic lobes, a single insertion line was chosen for further study (Villano, 1995). Similarly, in the Brodsky (1996) study, an enhancer trap screen was carried out for patterned expression patterns in the eye.

In the third instar larval leg disc, beta-gal expression marking transcription of fj is expressed in a pattern of concentric circles, similar to a subset of the concentric restrictions that mark the future segment boundaries of the leg. Non-uniform expression is observed in fj eye discs, with strongest expression in the central portion of the disc just anterior to the morphogenetic furrow, but with no expression in the lateral regions of the disc. An apparent gradient of expression is seen centrally declining toward the posterior tip of the disc. This triangular zone of expression does not correspond to any known developmental or physiological compartment boundaries. In the larval brain, expression is concentrated in the outer optic anlage (ooa), which forms a circumferential band around the optic lobe. The ooa is a proliferation zone that contributes cells to the lamina and distal medulla regions of the optic lobe, that form the postsynaptic targets for the photoreceptor axons. Staining in the eye and optic lobe are transient and no staining is detectable in adults. However, some beta-gal expression persists in the wings, legs and antennae of adult flies (Villano, 1995).

In the leg, segments occupying intermediate positions in the PD axis are specifically affected in the strongest mutations, fj2 and fj3. The adult leg can be divided into five regions. The most distal portion of the leg is called the tarsus, followed proximally by the tibia, femur, trochanter and coxa. The tarsus is composed of five segments, T5-T1 (distal to proximal), which are separated from each other by joints and are distinguishable by their lengths, patterns of bristles and cuticular specializations. The tarsus is reduced to four segments in these mutants. The middle tarsal segments, T2 and T3, are replaced by a single segment that appears to be a fusion of these two, while T1, the tibia and the femur are truncated. The more proximal segments of the leg (the coxa and trochanter) and the most distal (T5 and T4) are unaltered in fj mutants. Even within the affected segments, anterioposterior and dorsal-ventral patterning appears to be normal, based on cuticular patterns (Villano, 1995).

Loss of growth in the PD axis of the leg and wing might arise from an initial failure in proliferation and differentiation, or might result from a late degeneration event subsequent to proliferation. To distinguish between non-proliferation and degeneration, the emergence of the tarsal segments in prepupal discs that had just begun to evert was examined. By 4 hours APF in wild-type discs, all five of the prospective tarsal segment boundaries can be observed as indentations of the leg epithelium. Only four such boundaries are seen in fj2 discs and the sizes of the presumptive T2/T3 and T1 segments are already abnormally small (also see Waddington, 1943). Presence of cell death was examined by acridine orange staining during the early or late third instar, between the time of active cellular proliferation in the leg disc and the time of leg eversion. No cell death was found. These results suggest that the fj leg phenotype arises from an initial failure in the proliferation of a subset of tarsal segments, as well as a failure to initiate the T2/T3 segment boundary (Villano, 1995).

Previous work on the fj gene suggested that it functions as a regional signaling molecule regulating growth and differentiation in specific portions of the PD axis in the leg and wing. Analysis of the phenotype, expression pattern, molecular identity and biochemistry of fj strongly supports this hypothesis. A role in intercellular communication may be inferred from the nonautonomous behavior of wild-type cells juxtaposed with mutant cells in mosaic patches at the T2/T3 boundary in the leg (Tokunaga, 1976). The heterozygous cells immediately adjacent to the mutant patch fail to form a joint. This would be expected if a defective signal from the mutant cells was unable to stimulate a receptor on the immediately adjacent wild-type cells. However, the majority of fj clones crossing the T2/T3 segment boundary display autonomous failure to form a joint (the nonautonomous group were attributed to probable mitotic recombination between the marker and the fj gene), suggesting fj may be required in both cells. A nonautonomous signal or ligand would presumably be in the form of a cell surface or secreted molecule, although the molecule defective in the mosaics could also be involved in the generation of the signal. Analysis of the fj gene product is consistent with the fj protein representing the signal itself. Conceptual translation of fj cDNA predicts a type II trans-membrane glycoprotein with a potential internal signal peptidase cleavage site. In vitro translation of fj mRNA in the presence of membranes results in cleavage of a portion of the nascent polypeptides at the predicted site. However, the microsomal cleavage of fj is incomplete and a significant portion of the protein remains transmembrane and full length (Villano, 1995).

It is thought that fj is required for local proliferation in imaginal tissue. (1) Loss of function results in a loss of tissue in both the leg and the wing. In the leg, a loss of growth is apparent as early as the prepupa. (2) fj is strongly expressed in known proliferation zones, including the region of proliferation just anterior to the morphogenetic furrow of the eye disc and the outer proliferation center of the optic lobe. Although only mild phenotypes in the eye were found in the Villano (1995) study, Waddington (1943) observed that the eye is greatly diminished or lost in double mutants of fj and dachsous (ds), a gene with homology to cadherins that by itself has no phenotype in the eye. A role for fj in controlling growth of the eye may therefore be masked by the activity of other genes. (3) Two distinct tumor-suppressor genes, Gull, an allele of fat (Bryant, 1988 and Mahoney, 1991) and expanded (Boedigheimer, 1993), are able to suppress the fj leg phenotype in double mutants (Villano and Katz, unpublished observations cited in Villano, 1995), suggesting that loss of fj can be compensated for by deregulation of proliferative activity. Whether fj is sufficient for growth can be tested by ectopic expression. However, if fj is indeed a signal then the effect of ectopic expression will depend on the distribution of its receptor and downstream intracellular signaling pathways, which may not be ubiquitous (Villano, 1995).

Cleavage and secretion is not required for Four-jointed function in Drosophila patterning: fj 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).

Fj has been proposed to act as a secreted signalling molecule, based on the fact that its C-terminal region can be cleaved and secreted, and that it exhibits non-autonomous functions in mosaic clones. The functional significance of this cleavage was tested by making modified forms of Fj that are either poorly cleaved, constitutively secreted or anchored in the Golgi apparatus. Several different assays support the conclusion that cleavage and secretion of Fj is not essential for its activity in either planar polarity or PD patterning. In both overexpression experiments and rescue assays, Golgi-tethered Fj has significantly more activity than wild-type Fj, while secreted Fj is less active. Furthermore, even though Golgi-tethered Fj is not secreted, it still produces non-autonomous polarity phenotypes similar to or stronger than those of wild-type Fj. Therefore, it is proposed that secreted Fj is not the active form, and that Fj acts intracellularly. Fj most likely acts by modulating the activity of other molecules involved in intercellular signalling (Stutt, 2004).

Thus, the results show that rather than acting in an analogous manner to the cleaved type II transmembrane protein Hedgehog, a better model for Fj function may be the type II transmembrane protein Fringe (Fng). Fng is Golgi-localized and acts as a glycosyltransferase enzyme to post-translationally modify the receptor Notch (N). This renders N more sensitive to its ligand Delta, and less sensitive to the ligand Serrate. In the case of Fj, there are no molecular homologies that give any clues as to a possible enzymatic activity. Consequently, the precise location of its function is uncertain. However, since the results show that if Fj is tethered in the Golgi, it has higher than normal activity, the Golgi seems most likely to be its preferred site of action (Stutt, 2004).

An important question is whether Fj cleavage has any functional significance. Since forced retention of Fj in the Golgi causes hyperactivity, it is possible that cleavage and secretion could be a mechanism to downregulate Fj activity during normal development. However, further experiments will be required to determine if the cleavage step is temporally or spatially regulated (Stutt, 2004).

The mouse homolog of Fj (Fjx1) has also been proposed to act as a secreted molecule, on the basis of a hydrophobic stretch at the N terminus that might represent a signal peptide and the presence of predicted signal peptidase cleavage sites. However, the hydophobic region is not at the extreme N terminus and is sufficiently long to be predicted to be a transmembrane domain. This structure suggests that Fjx1 may also be a type II transmembrane protein. Consistent with this, in tissue culture experiments, no secretion of the C-terminal region of Fjx1 into the medium is observed. However, this failure to detect any activity of Fjx1 when overexpressed in flies suggests that there may be a divergence of function between the fly and vertebrate proteins (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).

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 (FjGGG; G, glycine). The expression levels and Golgi localization of FjGGG appear normal, but FjGGG 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 FjGGG. 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, Ds2-3S236A (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 (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 Ds2-3S236A 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 sFjGGG was used as the enzyme. Moreover, Ds2-3S236A 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:FjGGG, 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 (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 this 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 fjGGG 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 FjGGG: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 FjGGG: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).

The Dachsous/Fat/Four-Jointed pathway directs the uniform axial orientation of epithelial cells in the Drosophila abdomen

The achievement of the final form of an individual requires not only the control of cell size and differentiation but also integrative directional cues to instruct cell movements, positions, and orientations. In Drosophila, the adult epidermis of the abdomen is created de novo by histoblasts. As these expand and fuse, they uniformly orient along the anteroposterior axis. The Dachsous/Fat/Four-jointed (Ds/Ft/Fj) pathway is key for their alignment. The refinement of the tissue-wide expression of the atypical cadherins Ds and Ft result in their polarization and directional adhesiveness. Mechanistically, the axially oriented changes in histoblasts respond to the redesign of the epithelial field. It is suggested that the role of Ds/Ft/Fj in long-range oriented cell alignment is a general function and that the regulation of the expression of its components will be crucial in other morphogenetic models or during tissue repair (Mangione, 2018).

PCP pathways act as key coordinators in the makeover of planar epithelial tissues during development by modulating adhesive interactions and mechanical forces. However, the regulatory means that these pathways use to direct the topographical organization of epithelial cells are far from being clear. By applying in vivo analyses to the morphogenesis of the adult abdominal epidermis of Drosophila, this study has characterized a new mechanism for the stepwise long-range cobblestone organization of the tissue. The organization of the abdominal epithelial landscape was found to be the result of an axially oriented adhesiveness mediated by the Ds/Ft/Fj pathway. The directional cues dictated by this pathway put the epithelial cells on the right track, orienting their otherwise changing shapes along the A/P axis (Mangione, 2018).

Global tissue changes may involve many different activities: coordinated cell-cell rearrangements triggering tissue reorientation or convergent extension,or spatially controlled proliferation and growth, division orientation, and death. The uniform axially oriented alignment of histoblasts emerges in a precise spatiotemporal manner through coordinated changes in cell shape orientation. Histoblasts constitute a highly proliferative tissue with much room for expansion. In this scenario, which is very different from that of epithelial tissues constrained in their dimensions like the fly notum, to reach uniformity in cell alignment orientations, changes in cell shape and area would be preferred over cell intercalations. When the expressions of ds, ft, or fj are affected, the relative orientations of cell alignments are severely disturbed without alterations in tissue differentiation (Mangione, 2018).

It is not known which positional cues the Ds/Ft/Fj pathway interprets to dictate the stereotyped uniform anisotropic polarization of the abdominal epidermis along the A/P axis. Some tips may come from analyses on the regulation of the components of the core Fz-PCP system. This system is a required partner of the Ds/Ft/Fj pathway for proper planar polarity acquisition. Both pathways rely on primary long-range global cues to coordinate short-range cell polarity. During the development of the eye and wing in Drosophila, the secreted factor Wingless (Wg/Wnt1) modulates intercellular interactions in both systems. In the Ds/Ft/Fj pathway, Ds and Ft form heterodimers, whereas in the Fz-PCP system, there are Fz-Vang heterodimers and Stan-Stan homodimers. In the eye and the wing discs, Wg binds to Fz, which affects Fz-Vang interactions and the activity of the Fz-PCP system. As a result, cells orient toward the source of Wnt expression (Wg and Wnt4) at the compartment margins. Wg/Wnt4 could be playing an equivalent role during the axial uniform alignment of oriented cells in the abdomen. While Wg specifies the tergite and sternite cell fates, how it could regulate the graded expression of ds, ft, or fj or influence the uniform axial orientation of histoblasts remains poorly explored (Mangione, 2018).

It is known that differential adhesive properties between neighboring cells prevent intermingling, as they tend to minimize their contacts. In clones, this leads to smooth borders. Major differences were found in roughness, perimeter, and, to a lesser extent, roundness in mutant clones for members of the Ds/Ft/Fj pathway. These differences strongly support a role for the Ds/Ft/Fj pathway and, in particular, the opposing graded expressions of the atypical cadherins Fat and Ds, in generating directional information at cell contacts. Mutant clones generate planar conflicts between cells with different adhesive properties and smooth borders at specific edges, and the directional information is lost (Mangione, 2018).

The evolving functional pattern delineated by the Ds/Ft/Fj pathway arises as an elegant and efficient way to dictate directional order across developmental fields. Several pieces of evidence point to it as a key element modulating similar processes in different organisms. Mitral valve prolapse (MVP) is a common cardiac valve disease, the genetic etiology of which has remained elusive. Recently, it has been shown that MVP could be traced back, both in mice and fish models, to developmental errors in valve morphogenesis. The epicardial-derived cells fail to uniformly align into the posterior leaflet, and this failure correlates to a missense mutation in the DCHS1 gene, the human homolog of ds. Similarly, the establishment of polarized arrays of aligned chondrocytes in the zebrafish developing pharyngeal arch requires Fat3 and Dchs2 cadherins. During postnatal stages in mammals, hair follicles progressively get in line, both locally and globally, precisely with the A/P axis. This process of postnatal refinement has been suggested, although not proved, to be the result of instructive functions originating from the Ds/Ft/Fj pathway. Whether vertebrate Ds/Ft/Fj signaling is essential to propagate polarity at a distance, is linked to molecular gradients, or may interact with other polarizing signals remains unknown (Mangione, 2018).

The subcellular polarization of the atypical myosin Dachs (D) in response to the Ds/Ft/Fj pathway appears to be uncoupled from the uniform orientation of cell alignments; D polarity is reoriented but sustained in ds, and its loss did not affect cell alignments uniformity. These data led to a hypothesis that a bias in contractility mediated by D at the cell cortex may be not critical for uniform cell orientation. Moreover, contractile anisotropy as a factor directing the axial uniformity of histoblasts is unsupported by the observed isotropic distribution of vinculin at cell vertices. In this scenario, asymmetric adhesiveness through heterodimeric interactions between Ds and Ft appears to be a more plausible element directing the uniform orientation of histoblasts (Mangione, 2018).

Assuming that cells and tissues tend to minimize their surface free energy, contacts through adhesion molecules and contractile activities at the cell cortex would be key determinants of cell and tissue shape. Adhesiveness will promote cells to spread their shared surface, while contractility will counterbalance the adhesive forces. Differential adhesive properties within histoblasts would introduce anisotropic tension affecting cell-cell contacts and the capacity to coordinately orient shape changes in the tissue plane. This anisotropic tension, which is revealed by laser microsurgery, is lost in ds mutants. Anisotropic tension is also uncovered in clones, in which contact angles and lengths between histoblasts adjust to their conflicting genotypes and their relative location. Differential adhesiveness at cell junctions would have direct input into surface tension. Tensile patterns rather than cell positions would therefore play instructive roles in the acquisition of uniform order. At any given point in time, they will reflect the recent developmental history of the tissue. Along this line, during the expansion and remodeling of the histoblasts, the expression pattern of Ft modulated by Ds and fj evolves into an A/P gradient spanning whole compartments. This expression refinement will result in the spreading throughout the epithelium of a counterbalanced adhesion share between Ft and Ds that will delineate the axially oriented surface tension landscape that will instruct uniform cell alignments (Mangione, 2018).

Will the final arrangement of the cells be physically stable (minimal energy) upon completion or will a secondary event be necessary to stabilize it? In Caenorhabditis elegans, the epidermal cells elongate during development and subsequently attach to the cuticle to fix their shape. Thus, the collagenous exoskeleton secreted by the apical surface of the epidermis seems to be indispensable. The histoblasts use their apical surface to attach to the overlaying pupal cuticle very early on. These contacts are very dynamic during the period of expansion and become stabilized by the end of tissue remodeling. Whether they fulfill a hardening role on the tissue landscape is an open question (Mangione, 2018).

The role this study has uncovered for the Ds/Ft/Fj pathway implementing the uniform orientation of the alignment of histoblasts does not relate to any developmental function in patterning, cell specification, and/or differentiation. The directional evolution of the expression of the different Ds/Ft/Fj pathway elements sets a spatially and temporally controlled directional adhesive partnership between Ds and Ft. This provides the basis for the establishment of a dynamic adhesiveness pattern unfolding over time that directs cell shape changes, ultimately guiding the uniform alignment of the epithelial cells across the tissue (Mangione, 2018).


Transcriptional Regulation

Notch activation at the midline plays an essential role both in promoting the growth of the eye primordia and in regulating eye patterning. Specialized cells are established along the dorsal-ventral midline of the developing eye by Notch-mediated signaling between dorsal and ventral cells. D-V signaling in the eye shares many similarites with D-V signaling in the wing. In both cases an initial asymmetry is set up by Wingless expression. Both eye and wing cells then go through a distinct intermediate step: in the wing, Wingless represses the expression of Apterous, a positive regulator of fringe (fng) expression; in the eye, Wingless promotes the expression of mirror (mrr), which encodes a negative regulator of fringe (unpublished observations of McNeill, Chasen, Papayannopoulos, Irvine, and Simon, cited by Papayannopoulos, 1998). Both wing and eye cells share a Fng-Ser-Dl-Notch signaling cassette to effect signaling between dorsal and ventral cells and establish Notch activation along the D-V midline. Local activation of Notch leads to production of diffusible, long-range signals that direct growth and patterning, which in the wing include Wingless, but in the eye remain unknown. At least one downstream target of D-V midline signaling, four-jointed (fj), is also conserved. four-jointed is also expressed in the wing and its expression there is indirectly influenced by Notch (Papayannopoulos, 1998 and references).

During early eye development, fringe is expressed by ventral cells. This expression appears to be complementary to that of the dorsally expressed gene mrr. During early to mid-third instar, additional expression of fng appears in the posterior of the eye disc. This line of posterior fng expression is just in front of the morphogenetic furrow and moves across the eye ahead of the furrow. In the wing disc, Dl and Ser induce each other's expression, and become up-regulated along the D-V border where they can productively signal. Dl and Ser are also preferentially expressed along the D-V midline during eye development. Ser expression, like fng expression, is complementary to that of mrr, whereas Dl expression partially overlaps that of mrr. The spatial relations among fng, Ser, and Dl expression in the eye are thus similar to those in the wing, although in the wing, their expressions are inverted with respect to the D-V axis (Papayannopoulos, 1998).

The four-jointed gene is expressed in a gradient during early eye development, with a peak of expression along the D-V midline. Together with Ser and Dl, Fj serves as a molecular marker of midline fate. Ubiquitous expression of Fng during early eye development, generated by placing fng under the control of an eyeless enhancer, eliminates detectable expression of Ser and Dl along the midline. Conversely, misexpression of Fng in clones of cells, can result in ectopic expression of Ser and fj that is centered along novel borders of Fng expression in the dorsal eye. Ectopic Ser and fj expression can also be detected along the borders of fng mutant clones in the ventral eye. These observations show that Fng expression borders play an essential and instructive role in establishing a distinct group of cells along the D-V midline of the developing eye. Animals with reduced fng activity have small eyes. Moreover, ubiquitous fng expression also results in a dramatic loss of tissue. Tissue loss is detectable in the developing imaginal disc, before the morphogenetic furrow moves across the eye. Moreover, eye loss is observed when fng is ectopically expressed during early development, but not when fng is ectopically expressed behind the furrow. These observations indictate that a Fng expression border is required for eye growth, specifically during early eye development (Papayannopoulos, 1998).

Fng differentially modulates the action of Notch ligands in the eye just as it does in the wing. Clones of cells ectopically expressing Dl can induce Ser expression in ventral, Fng-expressing cells, but not in dorsal cells. Fng alone can induce Ser expression in dorsal cells, but only near the D-V midline. When Fng and Dl are co-misexpressed, Ser expression can be induced in dorsal cells even when the clones are far from the D-V midline. Clones of cells ectopically expressing Ser are able to induce increased expression of Dl in dorsal cells but not in ventral, Fng expressing cells. However, if Ser is ectopicallly expressed in fng mutant animals, it can induce Dl expression in ventral cells (Papayannopoulos, 1998).

Notch function is also necessary for normal D-V midline cell fate. The ability of Ser and Dl to induce one another's expression indicates that the expression of either one is a marker for Notch activation in the eye. Analysis of loss-of-function mutants of Notch and its ligands, as well as ectopic expression studies, indicate that Notch activation also regulates eye growth. Several observations indicate that the D-V midline is the focus of Notch activation required for growth. Moreover, the midline corresponds to a fng expression border, which is essential for growth and modulates Notch signaling during early eye development. Because local activation of Notch has long-range effects on growth and four-jointed expression, it is inferred that Notch induces the expression of a diffusible growth factor at the midline. Notch activation influences ommatidial chirality. fng mutant clone borders within the ventral eye can be associated with reversals of ommatidial chirality, whereas mutant clones that cross the D-V midline disrupt the normal equator. The equatorial bias in the influence of ectopic Notch activation implies that the equator is the normal source of a Notch-dependent, chirality-determining signal (Papayannopoulos, 1998).

The Drosophila eye is composed of about 800 ommatidia, each of which becomes dorsoventrally polarised in a process requiring signaling through the Notch, JAK/STAT and Wingless pathways. These three pathways are thought to act by setting up a gradient of a signaling molecule (or molecules) often referred to as the 'second signal'. Thus far, no candidate for a second signal has been identified. The four-jointed locus encodes a type II transmembrane protein that is expressed in a dorsoventral gradient in the developing eye disc. The function and regulation of four-jointed (fj) during eye patterning has been analyzed. Loss-of-function clones or ectopic expression of four-jointed results in strong non-autonomous defects in ommatidial polarity on the dorsoventral axis. Ectopic expression experiments indicate that localized four-jointed expression is required at the time during development when ommatidial polarity is being determined. In contrast, complete removal of four-jointed function results in only a mild ommatidial polarity defect. four-jointed expression has been found to be regulated by the Notch, JAK/STAT and Wingless pathways, consistent with it mediating their effects on ommatidial polarity. It is concluded that the clonal phenotypes, time of requirement and regulation of four-jointed are consistent with it acting in ommatidial polarity determination as a second signal downstream of Notch, JAK/STAT and Wingless. Interestingly, it appears to act redundantly with unknown factors in this process, providing an explanation for the previous failure to identify a second signal (Zeidler, 1999).

Both in situ hybridization for fj transcripts and the lacZ activity patterns revealed by enhancer traps in the fj locus indicate that fj is normally expressed most strongly in a broad domain around the dorsoventral midline of the eye imaginal disc). To determine whether this localized expression is functionally significant, fj was ectopically expressed during eye development. Ectopic expression of fj was driven at the poles of the eye during eye patterning using an optomotor-blind driver. This results in dorsoventral inversions of ommatidial polarity at both the dorsal and ventral poles of the eye, often with three or more rows of ommatidia inverted (Zeidler, 1999).

The expression pattern of fj, and the phenotypes that were observed for loss-of-function and gain-of-function of fj activity, indicate a role for fj function in ommatidial polarity determination along the dorsoventral axis. Recent studies have revealed functions for the N, JAK/STAT and Wg pathways as regulators of ommatidial polarity determination, with the current model suggesting that Notch and Upd are positive regulators of a graded signal that is highest at the equator, whereas Wg is a negative regulator of such a factor (or factors). The fj gene is therefore a good candidate for being a downstream target of regulation by one or more of these pathways. Consistent with this, fj is regulated by the JAK/STAT and Wg pathways. In clones mutant for the Drosophila JAK homolog hop, which lack JAK function, a reduction in fj expression is observed. Although JAK is a cell-autonomously acting signal-transduction component, the effect on fj expression is not cell-autonomous, with greatest downregulation being observed in the center of the clone. In accordance with downregulation in hop clones, clones of cells ectopically expressing the JAK ligand Upd result in activation of fj expression. Conversely, ectopic expression of Wg (which is predicted to be a negative regulator) results in downregulation of fj expression. Activated N can nonautonomously activate fj expression. Taken together, these results indicate that fj is regulated by all three of these pathways in a manner consistent with mediating their functions in dorsoventral polarity determination (Zeidler, 1999).

One of the noteworthy aspects of fj regulation by the Notch and JAK/STAT pathways is that it is non-autonomous, even when it is studied using cell-autonomously acting signaling components such as the intracellular domain of N, Nintra. One possible explanation for this non-autonomy would be that fj is able to activate its own expression via an autoregulatory loop. To test this hypothesis, fj was ectopically expressed in the presence of a fj enhancer trap and it was found that fj was indeed able to activate its own expression. The activation of fj expression by ectopic expression of fj is non-autonomous, again consistent with the proposed secreted nature of the fj gene product. In addition to the N, JAK/STAT and Wg pathways, the only other gene reported to non-autonomously influence ommatidial polarity is frizzled (fz). A possible mechanism for non-autonomy of fz function would be via regulation of fj expression. The expression of fj was examined in fz loss-of-function clones and in clones of cells ectopically expressing fz, but in neither case is there any change in fj expression (Zeidler, 1999).

Protein Interactions

Planar cell polarity in the Drosophila eye is directed by graded Four-jointed and Dachsous expression

Planar cell polarity (PCP) occurs when the cells of an epithelium are polarized along a common axis lying in the epithelial plane. During the development of PCP, cells respond to long-range directional signals that specify the axis of polarization. It has been proposed that with respect to Drosophila eye morphogenesis a crucial step in this process is the establishment of graded expression of the cadherin Dachsous (Ds) and the Golgi-associated protein Four-jointed (Fj). These gradients have been proposed to specify the direction of polarization by producing an activity gradient of the cadherin Fat within each ommatidium. In this report, the key predictions of this model were tested and confirmed by altering the patterns of Fj, Ds and Fat expression. It was shown that the gradients of Fj and Ds expression provide partially redundant positional information essential for specifying the polarization axis. It was further demonstrated that reversing the Fj and Ds gradients can lead to reversal of the axis of polarization. Finally, it was shown that an ectopic gradient of Fat expression can re-orient PCP in the eye. In contrast to the eye, the endogenous gradients of Fj and Ds expression do not play a major role in directing PCP in the wing. Thus, this study reveals that the two tissues use different strategies to orient their PCP (Simon, 2004).

The development of organized PCP requires cells to polarize in response to directional signals within the plane of the epithelium. The apparent absence of local cues has suggested that cells orient their polarity in response to long-range diffusible signaling molecules that form gradients across the tissue. It has been proposed that the role of the diffusible signals, such as Wingless produced at the poles of the eye disc, is to drive graded transcription of Ds and Fj. In this model, the resulting Ds and Fj protein gradients then regulate the function of the cadherin Ft, resulting in a Ft activity gradient, which in turn controls the pattern of Fz competition within each ommatidium. Crucial tests of the model have been precluded by an inability to alter the patterns of Ds and Ft expression. This study has analyzed the effects of altering Fj, Ds and Ft expression in the eye, and provides evidence supporting crucial features of the model. Most importantly, it has been demonstrated that the Fj and Ds expression gradients provide redundant directional information that together orient PCP. Furthermore, the data shows that it is the combination of both gradients that provides the robust directional cues needed to support the perfect fidelity of polarization in wild-type eyes. In addition, it has been shown that graded Ft expression can direct the pattern of ommatidial polarity, thus providing support for the role of Ft as a graded regulator of Fz signaling acting under the control of the Fj and Ds gradients (Simon, 2004).

In the proposed model, the consistent equatorial bias of Fz signaling results from more effective Ft action in each equatorial R3/4 precursor cell when compared with its adjacent polar counterpart. Since this Ft difference results from the action of the Fj and Ds gradients, a key question is how these gradients could control the level of Ft function. Important insight into this issue has come from studies of the wing that suggest that Ft and Ds form a complex in which the localization of Ft on the surface of one cell is promoted by binding to Ds on the surface of the neighboring cell. The dependence of Ft plasma membrane localization on Ds may account for the requirement for Ds function during planar polarization in the eye, even when sufficient directional cues are provided by the Fj expression gradient (Simon, 2004).

The existence of Ds:Ft intercellular dimers suggests several mechanisms by which Ds might regulate Ft. One simple possibility is that Ds merely controls the accumulation of Ft on the surface of the neighboring cell. Thus, the relatively higher level of Ds in the polar R3/R4 precursor, which results from the polar gradient of Ds expression, would lead to the accumulation of more Ft on the bordering surface of the equatorial cell. This would result in an asymmetry in Ft protein levels precisely along the border between the precursor cells where Fz/PCP competition occurs. Although no such gradient has been observed, it would certainly be very subtle and perhaps undetectable. A second possibility is that Ds binding to Ft regulates Ft activity rather than localization. A third possibility is that Ds could participate with Ft in binding to the extracellular domain of a downstream target (Simon, 2004).

Fj appears to play a more limited role than Ds during planar polarization of the eye. Unlike Ds, which both contributes a directional signal through its graded expression and plays an essential role in the interpretation of directional cues, Fj appears only to participate in PCP establishment via the directional information provided by its graded expression. This more limited role can be seen in the observations that either the absence or the ubiquitous expression of Fj yields equivalent phenotypes, and does not grossly disrupt the pattern of polarization unless the Ds gradient has been replaced with ubiquitous expression. How might graded Fj fulfill this role? One possibility is that Fj may regulate the ability of Ft and Ds to productively interact with each other. Thus, the higher expression of Fj in the equatorial cell of each ommatidium leads to more Ft:Ds dimers being formed with Ft in the equatorial cell than in the opposite orientation. Since Fj appears to function in the Golgi, this regulation may involve the direct modification of Ft or Ds (Simon, 2004).

It is important to note that one aspect of the data reported here requires reconsideration of a feature of a previous model. In previous work, it was proposed that Fj acts upstream of Ds, perhaps by modifying the Ds activity gradient. This placement was based on genetic experiments showing that strong differences in Fj activity between R3/R4 precursor cells can direct ommatidial polarization only when Ds is present. The identification of an essential gradient-independent function for Ds clearly complicates the interpretation of these epistasis experiments. As a result, it is no longer possible to infer whether the information provided by the Fj expression gradient acts upstream of Ds to modify the information provided by the Ds gradient. An equally plausible possibility is that Fj regulates the function of the Ds:Ft complexes by modifying Ft rather than Ds function (Simon, 2004).

The work presented here was designed to test specific predictions of the model proposed in an earlier study. However, alternate roles for Ft function have also been proposed. In one model, Ft regulates the production of an unidentified long-range signal that is secreted at the equator and that directly controls eye polarity. The existence of such an unidentified patterning signal, often called Factor X, has been invoked frequently to explain the 'domineering nonautonomy' phenomenon seen in both the wing and the eye near clones of cells lacking function of PCP genes such as Fz. In the alternate model, the role of Ft is to prevent production of this factor everywhere in the eye except at the equator where Ft activity is proposed to be inhibited by unspecified mechanisms, presumably involving Ds. An important distinction between the two models relates to the predicted effects of graded Ft expression. In the model, graded Ft activity provides the key PCP directional cues, and thus ectopic Ft expression gradients are predicted to have the potential to orient ommatidial polarity. In an alternate model, gradients of Ft activity do not provide directional cues. Instead, it is the lack of Ft activity in a sharp zone at the equator that leads to the production of the unidentified patterning factor. As a result, this second model predicts that subtle gradients of Ft expression should not orient polarity, especially in the polar regions of the eye where Ft activity is proposed to be uninhibited. Thus, the data presented in this report demonstrating the orienting ability of Ft expression gradients presents a challenge to this alternate model. In addition, the need for Factor X, whose putative existence has been a common feature of PCP models in both the wing and eye, has been challenged recently on both experimental and theoretical grounds. These reports suggested that domineering nonautonomy results from the tendency of neighboring cells to align their polarization rather than the existence of an additional polarizing signal (Simon, 2004).

The key roles of Ft and the Fj and Ds expression gradients in the eye naturally raised the question of whether similar mechanisms are used to provide directional cues in other tissues, such as the wing. That such conservation might exist was suggested by the existence of gradients of Fj and Ds in the wing. Additionally, it has been demonstrated recently that ectopic gradients of Ft and Ds expression in the wing can produce re-orientation of polarity in the wing. Given the redundant nature of the directional cues provided by the Fj and Ds gradients in the eye, the most rigorous way to evaluate the roles of the Ds and Fj expression gradients in the wing was to examine the consequences of removing the directional information of both gradients simultaneously. When this was done, the resulting wings displayed almost completely normal polarity. Thus, the Ds and Fj expression gradients do not play a major role in orienting PCP in most of the wing blade. One possibility is that there are additional directional signals that act redundantly with the Ds and Fj gradients. Another possibility is that these gradients exist for reasons unrelated to PCP. For example, they may serve to regulate the function of Ft as a regulator of cellular proliferation. Possible support for such a role comes from the observation that flies in which both graded Fj and Ds expression has been replaced with ubiquitous expression survive to adulthood at reduced frequencies, and often display defects in the size and shape of their legs, wings and eyes (Simon, 2004).

The dispensability of the Fj and Ds gradients of expression during the polarization of the wing indicates that there must be currently unidentified directional cues directing wing PCP. Despite their mysterious nature, it is likely that their mode of action will involve the Ds:Ft complex. This inference can be drawn from the observation that animals lacking Ds function, or clones of cells lacking Ft or Ds activity, have substantial PCP defects in the wing. Importantly, clones of ft mutant cells in the wing appear not to read directional cues and instead align their polarity with that of their neighbors. Thus, whatever the nature of the unidentified signals, they appear not to function effectively in the absence of Ds and Ft. Since neither Ft nor Ds is directly required for the Fz PCP signaling at cell-cell junctions, the dependence of these unidentified signals on Ds and Ft suggests that they may act by asymmetrically modifying the action of the Ds:Ft complexes at cell-cell junctions engaged in PCP signaling. Thus, the elegant regulation of polarity in the eye by graded Fj and Ds expression may represent only one of a number of ways to modulate the action of Ft. Further analysis of the mechanisms by which Ft and Ds regulate the pattern of Fz/PCP signaling will undoubtedly aid in the identification of these unknown signals and their mode of action (Simon, 2004).

Planar polarity specification through asymmetric subcellular localization of Fat and Dachsous

Two pathways regulate planar polarity: the core proteins (Warts-Hippo) and the Fat-Dachsous-Four-jointed (Ft-Ds-Fj) system. Morphogens specify complementary expression patterns of Ds and Fj that potentially act as polarizing cues. It has been suggested that Ft-Ds-Fj-mediated cues are weak and that the core proteins amplify them. Another view is that the two pathways act independently to generate and propagate polarity: if correct, this raises the question of how gradients of Ft and Ds expression or activity might be interpreted to provide strong cellular polarizing cues and how such cues are propagated from cell to cell. This study demonstrates that the complementary expression of Ds and Fj results in biased Ft and Ds protein distribution across cells, with Ft and Ds accumulating on opposite edges. Furthermore, boundaries of Ft and Ds expression result in subcellular asymmetries in protein distribution that are transmitted to neighboring cells, and asymmetric Ds localization results in a corresponding asymmetric distribution of the myosin Dachs. The generation of subcellular asymmetries of Ft and Ds and the core proteins is largely independent in the wing disc, and additionally ommatidial polarity in the eye can be determined without input from the Ft-Ds-Fj system, consistent with the two pathways acting in parallel (Brittle, 2012).

The results demonstrate the importance of gradients and boundaries of Ds and Fj expression in the generation of cellular asymmetry. Previous reports have suggested that weak differences in Ft and Ds binding across cells could be amplified to produce asymmetric localization of downstream pathway effectors such as Dachs. This study reports significant asymmetry of both Ft and Ds localization, suggesting that physical polarization of these proteins is an important part of the mechanism by which Ft-Ds-Fj generate polarity. This study thus reveals the Ft-Ds-Fj system as a mechanism for converting long-range morphogen-induced gene expression patterns into planar polarity cues at the level of individual cells (Brittle, 2012).

In the wing disc, Dachs asymmetry is particularly prominent at the pouch-hinge boundary where a strong disparity in Ds levels exists. In this situation, the Ds boundary may contribute to the high level of asymmetry, for instance, via a feed-forward mechanism that suggests that Dachs asymmetry is produced by strong differences in Ds and Ft binding between neighboring cells, that is passed from cell to cell as the wing grows. However, strong asymmetry of Ds and Dachs were also detected in the eye disc, where there is no evidence for sharp disparities of Ds or Fj, consistent with expression gradients providing sufficient cues. Dachs asymmetry was also seen in 6 hr pupal wings consistent with Ft-Ds-Fj signaling continuing to provide polarizing cues after the third-instar stage (Brittle, 2012).

The ability of shallow expression gradients to produce observable asymmetry of Ft and Ds distribution is unexpected. A possible mechanism is that a weak asymmetry in activity or protein distribution across the cell is amplified by a feedback loop to produce an observable protein asymmetry, in a manner similar to that suggested for the generation of core protein asymmetry. Notably, Dachs does not seem to be part of any such amplification mechanism. Indeed loss of Dachs activity appears to promote Ft and Ds asymmetry. It may be that cell divisions, which are reduced in dachs mutants, disrupt the appearance of asymmetry, possibly explaining the high level of variance of asymmetry of Dachs, Ft and Ds in WT tissue. To understand further how the asymmetry of Ft and Ds is achieved, and whether this requires an amplification mechanism, it will be necessary to combine more detailed quantitative analyses together with computational approaches (Brittle, 2012).

The data suggest that Ft and Ds asymmetry leads directly to the observed Dachs asymmetry in both wing and eye discs. Although no direct interactions were detected between Ds and Dachs, the colocalization and the similar degree of subcellular asymmetry observed for these proteins support a model in which Ds recruits Dachs (Brittle, 2012).

Finally, this study reassessed the link between Ft-Ds-Fj and the core planar polarity proteins. In the wing, it was demonstrated that throughout much of the third-instar disc, both Ft-Ds-Dachs and the core proteins independently adopt PD-oriented subcellular localizations, most likely under the influence of the morphogen gradients that pattern the axes of the tissue. However, in the most proximal regions of the wing (adjacent to the pouch-hinge boundary in the disc), Ft-Ds-Fj appear to act via Dachs to ensure correct polarization of the core proteins. The mechanism behind Dachs regulation of the core needs further investigation, but because Dachs plays a role in orientated cell division and influences apicolateral junctional length [28], these factors may be involved (Brittle, 2012).

In the eye, Ft-Ds-Fj seem to play a more general role in polarizing the core proteins throughout the tissue, apparently independently of Dachs activity. Ft-Ds-Fj may also provide a Dachs-independent input to the core in the wing, but data presented in this study suggest that it is at best redundant. Even in the eye, Ft-Ds-Fj are not absolutely essential for the core to polarize, indicating that there are other unknown inputs (Brittle, 2012).

An important observation is that complete loss of ft or ds activity in the eye or wing results in very strong defects in core protein polarity, but when overgrowth is suppressed in these backgrounds via manipulation of Wts-Hpo pathway activity, then much milder defects are observed. On one hand, excessive cell division alone may disrupt the process of planar polarity establishment by the core proteins, possibly due to asymmetric localization being lost each time a cell undergoes mitosis. Alternatively, Ft-Ds-Fj-mediated polarity cues may constitute more important inputs to the core proteins in proliferating tissues. Finally, it is possible that other Wts-Hpo pathway transcriptional targets, not related to growth, contribute to the planar polarity phenotype (Brittle, 2012).

Overall, the data support a model in which the Ft-Ds-Fj system and core planar polarity proteins act independently to generate and propagate planar polarity through the asymmetric subcellular distribution of their protein components. No evidence was found that the core proteins can influence the asymmetry of the Ft-Ds-Fj system; however, in particular contexts, the Ft-Ds-Fj system can act through different effectors to influence core protein polarity (Brittle, 2012).


In the third instar larval leg disc, beta-gal, marking the activity of four-jointed transcription, is expressed in a pattern of concentric circles, similar to a subset of the concentric restrictions that mark the future segment boundaries of the leg. While these patterns tend to overlap in the third instar leg, the localization of this expression is more clearly seen in prepupal leg discs 4-6 hours after puparium formation. At this time, eversion of the leg from the folded disc epithelium has just occurred and invaginations marking the future tarsal segment boundaries are clearly visible. A line of positive cells is seen adjacent to each tarsal furrow, with additional strong expression in the first tarsal segment and in the distal tibia. A weaker pattern of semicircular arcs is seen in the antennal discs at the third instar. Such expression patterns are predicted for genes involved in setting or reading values along the PD axis of the leg and suggests this gene might have such a function (Villano, 1995).

The four-jointed (fj) gene encodes a type 2 transmembrane protein and is also expressed in concentric rings within the developing leg imaginal disc. In fj mutants, growth of the femur, tibia, and first three tarsal segments is reduced, and the ta2-ta3 segment border is absent. The rings of fj expression in leg imaginal discs are complementary to the rings of Notch expression. Consistent with this complementarity, fj expression is inhibited in cells expressing activated Notch; in cells neighboring ectopically expressing Ser or Dl, and in cells along the borders of ectopic fng expression. By contrast, fj expression is activated within cells expressing Ser or Dl. These observations indicate that fj is negatively regulated downstream of Notch signaling in the leg. Thus, Notch signaling subdivides each leg segment into distinct domains of gene expression (Rauskolb, 1999).

Expression of beta-gal in the wing disc is concentrated in the prospective wing blade region, the most distal region of the disc. The beta-gal expression patterns in the visual system were unexpected and very unusual. The eye disc is composed of an epithelial sheet interrupted by a moving invagination, the morphogenetic furrow, which progresses from the posterior to the anterior edge of the disc during development. Cells in front of the furrow are unpatterned and actively dividing, while cells immediately posterior to the furrow become recruited into the differentiated clusters that will form the ommatidia of the adult eye. All genes that have been previously characterized in eye development are expressed uniformly across the width of the eye disc (in parallel to the morphogenetic furrow), reflecting the uniform requirements for photoreceptor and support cell differentiation in each of the repeating units. However, non-uniform expression is observed in fj eye discs, with strongest expression in the central portion of the disc just anterior to the morphogenetic furrow, but with no expression in the lateral regions of the disc. An apparent gradient of expression is seen centrally declining toward the posterior tip of the disc. This triangular zone of expression does not correspond to any known developmental or physiological compartment boundaries (Villano, 1995 and Brodsky, 1996).

Finally, in the larval brain expression is concentrated in the outer optic anlage (ooa), which forms a circumferential band around the optic lobe. The ooa is a proliferation zone that contributes cells to the lamina and distal medulla regions of the optic lobe, that form the postsynaptic targets for the photoreceptor axons. beta-gal expression is confined to the ventral portion of this band and only to the part that gives rise to the medulla. Expression diminishes distal to proximal in this zone. A single line of cells continued dorsally along a furrow that separates the lamina from the medulla-contributing portions of the ooa. In a screen of 5000 enhancer trap lines, this was the only gene that was recovered with asymmetric expression in the optic lobes. Such asymmetric distribution suggests a compartment that has not been previously described but which is predicted from work that suggests that the dorsal and ventral halves of the lamina and medulla (which arise from the ooa) are distinguishable by the axons of the photoreceptor neurons. Staining in the eye and optic lobe are transient and no staining has been detectable in adults. However, some beta-gal expression persists in the wings, legs and antennae of adult flies (Villano, 1995).

Given the role of fj in the establishment of polarity in the Drosophila eye and the significance of expression gradients for the establishment of planar polarity, fj expression was investigated in the developing wing using the enhancer detector fjP1, which accurately mirrors the pattern of FJ mRNA expression in vivo. While fj expression in the third-instar wing imaginal disc is confined to the wing pouch the pattern of expression shown by the enhancer detector at 6 h after prepupal formation (APF) is already graded with lower levels present in proximal regions. The graded expression is retained at subsequent stages, as detected both with anti-beta-galactosidase antibodies and by LacZ activity staining. Although there is no antibody available against the fj protein, the fidelity of the fj-lacZ reporter suggests that fj transcripts (and by implication Fj protein) are present in a gradient in the developing pupal wing. In contrast to the pattern seen in the wing, the highest levels of fj enhancer trap expression present in the abdominal segments of young adults are apparently limited to two transverse bands of small cells located across the a3 and p1 regions of each segment. While these bands represent the highest levels of staining at this time, more heavily stained specimens also indicate that a lower level of uniform expression is also present superimposed on the a3/p1 pattern. While intriguing, it should be noted that planar polarity has already been established by the time abdomens can be stained and the pattern seen may not mirror fj expression present during polarity establishment (Zeidler, 2000).

Gain-of-functions studies of four-jointed in the leg

The molecular basis of segmentation and regional growth during morphogenesis of Drosophila legs is poorly understood. four-jointed is not only required for these processes, but also can direct ectopic growth and joint initiation when its normal pattern of expression is disturbed. These effects are non-autonomous, consistent with the demonstration of both transmembrane and secreted forms of the protein in vivo. The similarities between four-jointed and Notch phenotypes led to an investigation of the relationships between these pathways. Surprisingly, it was found that although four-jointed expression is regulated downstream of Notch activation, four-jointed can induce expression of the Notch ligands, Serrate and Delta, and may thereby participate in a feedback loop with the Notch signaling pathway. four-jointed interacts with abelson, enabled and dachs, which suggests that one target of four-jointed signaling is the actin cytoskeleton. Thus, four-jointed may bridge the gap between the signals that direct morphogenesis and those that carry it out (Buckles, 2001).

To understand Fj function, whether Fj exists in vivo as a secreted or a transmembrane protein was investigated. Drosophila S2 cell lines containing the fj cDNA under the control of a heat-shock promoter were generated (S2:fj). Homogenates from these cells and from the parent S2 line were analyzed on Western blots using antibodies generated against Fj. Antisera detected three major bands that were present in the S2:fj cells after heat-shock but were absent from the S2 parent line. In cell fractionation experiments of the S2:fj cells, the two more slowly migrating forms behaved as integral membrane proteins, since they segregate with both the heavy and the light membrane fractions and can not be washed off the membranes by alkaline treatment. Their sizes are consistent with the full-length protein predicted from sequence analysis. By contrast, the smallest polypeptide is secreted into the medium and co-migrates on SDS-PAGE with the intact C-terminal domain (Buckles, 2001).

To detect Fj reliably in larval tissue, glycoproteins were first concentrated by precipitation with ConA Sepharose beads before loading on the gel for Western blot analysis. Under these conditions, three polypeptides of the same size seen in cultured cells were also detected in homogenates from wild-type third instar larvae but were absent from homogenates produced from larvae homozygous for mutant alleles of fj. Just as the in vitro forms were shown to be altered by endoglycosidase H treatment and therefore N-glycosylated, so all three larval forms can be bound by ConA and therefore contain asparagine-linked core glycosylation. While the relative abundance of the three forms is somewhat variable, under all conditions the largest transmembrane form is present in equal or up to five-fold excess of the secreted form. Thus a significant fraction of Fj protein remains membrane-bound, although some protein molecules are cleaved and secreted (Buckles, 2001).

Fj is expressed in a series of concentric rings in the developing leg and its expression in the tarsus is tightly associated with forming segment boundaries. Fj is necessary for the formation of the T2/3 joint and is required for growth of the femur, tibia and tarsal segments 1-3. If Fj is a key regulator of these morphogenetic processes, it might be expected that Fj would also be sufficient to initiate joint formation and growth in the leg. Furthermore, since fj is expressed in only a subset of the cells within each leg segment, whether distinct domains of fj expression are indeed important for proper leg development was investigated (Buckles, 2001).

Ubiquitous expression of fj in the developing legs was accomplished by either inducing HS-fj or by driving UAS-fj with 69B-Gal4. Ectopic fj expression is capable of inducing the formation of ectopic joint-like structures, which resemble the partial joints seen at T2/3 in fj hypomorphic alleles. In flies homozygous for the HS-fj insertion, 80.5% of legs contained such ectopic partial joints. These appeared as donut-shaped invaginations in T3 or, much less frequently, in T2 and T4, usually centered approximately equidistant from the flanking joints. These invaginations resemble the ball-and-socket structure of a normal tarsal joint and, like normal joints, contain only bare cuticle. In the HS-fj flies, these ectopic structures occur with minimal loss of growth in the segment and no loss of endogenous joints. These results suggest that fj is sufficient to initiate joint formation in the tarsus and that this capacity is largely independent of growth control in the segment. The UAS-fj driven expression causes more widespread ectopic joint-like structures in the tarsus, additional loss of PD growth within the tarsal segments, and loss of the T2/3 joint (Buckles, 2001).

Patterned misexpression of fj across multiple segment boundaries causes more dramatic results. Expression of fj along the AP compartment boundary using UAS-fj and ptc-Gal4 results in legs with reduced leg growth and segmental fusions. The effects are particularly dramatic in the tarsus. A similar effect is seen when dpp-Gal4 is used as the driver. In addition, these animals display occasional outgrowths from the leg. Some of these outgrowths appear to be segmented and most contain at least some bristles. Similar outgrowths and truncations are seen when expression is driven with en-Gal4, which is expressed within the posterior compartment of the leg disc. Together, these results suggest that the endogenous pattern of fj expression is critical to its function in both growth control and segmentation of the leg (Buckles, 2001).

Smaller, randomly positioned clones ectopically expressing fj were examined to address whether fj affects leg growth and segmentation non-autonomously, as would be predicted if Fj acts as a signaling molecule. Smaller patches of fj-expressing cells were produced using the flip-out actin-Gal4 technique, and such clones were marked by the cuticular marker yellow. Clones expressing fj that span a segment border result in the fusion of most segments. Effects are most dramatic in the tarsus, with a fusion of tarsal segments and accompanying reduced growth. Although in all examples an autonomous influence of fj was observed, in many instances apparent non-autonomy, in which joint structures were lost both within and adjacent to the clone, were observed (Buckles, 2001).

In addition to the disruptions in leg segmentation and growth observed with larger clones, occasional outgrowths from the leg were found. Importantly, these outgrowths were entirely composed of wild-type tissue, while the fj-expressing clone neighbored the outgrowth. Together, these results strongly argue that fj is a key regulator of leg segmentation and growth, and that fj can function non-autonomously in these processes (Buckles, 2001).

Fj is regulated downstream of N signaling and many of the phenotypes observed with ectopic fj expression are similar to those seen upon ectopic activation of N. It is possible that these similarities might derive from a common molecular cause. For example, deregulation of N signaling may cause a deregulation of fj expression, which would then disrupt normal morphogenesis. Alternatively, since fj is known to have a transcriptional feedback on its own expression, perhaps it also participates in a feedback loop onto the N pathway such that misexpression of fj actually results in misexpression of activated N. The most likely target for such feedback would be the N ligands, since N is expressed widely in the disc but only becomes activated at the restricted positions of ligand expression (Buckles, 2001).

To investigate whether Fj feeds back onto the N signaling pathway, the expression of the N ligands Ser and Dl was examined in leg discs in which fj was ectopically expressed along the AP axis using ptc-Gal4-driven expression of UAS-fj. Such misexpression of fj results in severe truncation of the tarsus. ptc is expressed at highest levels along the AP boundary, with graded expression in the anterior compartment of the disc. Ectopic fj expression induces the expression of both Ser and Dl along the posterior edge of the fj-expressing stripe, and does so largely non-autonomously. The non-autonomy is consistent with biochemical data, and provides further evidence that Fj acts as a signaling molecule. Furthermore, the ectopic expression of Ser and Dl, leading to ectopic activation of N, could account for some of the observed effects of ectopic fj expression on leg development (Buckles, 2001).

The asymmetry of induction only along the border of highest expression raised the possibility that induction might only occur at sharp boundaries of expression, such as that on the posterior edge of the ptc domain. To test this, UAS-fj was expressed with two additional drivers, dpp-Gal4 and en-Gal4, which are both expressed at somewhat lower levels than ptc-Gal4. dpp-Gal4 is expressed within the anterior compartment of the leg disc, while en-Gal4 is expressed in the posterior compartment with a sharp boundary of expression along the AP border. Misexpression of fj under either driver produces truncations of the tarsus as well as apparent outgrowths and/or bifurcations of the distal leg. As with ptc-Gal4, both dpp- and en-Gal4-driven expression of fj induces expression of Ser in cells neighboring those expressing high levels of fj: at the posterior edge of the dpp domain and at the anterior edge of the en domain. Similar non-autonomous induction of Dl is observed with these drivers (Buckles, 2001).

Whether fj is required for normal Ser expression was investigated. Ser expression was examined in pupal leg discs homozygous mutant for fj. Expression of Ser is unaffected in all leg segments except for one: Ser expression is significantly reduced in the second tarsal segment. This finding is consistent with the observation that fj mutants have a partial or complete lack of the joint between the second and third tarsal segments and reduced growth within the fused segment (Buckles, 2001).

Since fj induces Ser expression non-autonomously, it was of interest to examine their endogenous expression patterns during development of the leg. Consistent with the inductive behavior observed, fj and Ser appear to be expressed in adjacent but largely non-overlapping stripes in tarsal segments 2-4 in the developing leg disc (Buckles, 2001).

Together, these results suggest that there is a feedback loop between N ligand expression and the N target gene fj. Fj appears to be necessary for the initiation, upregulation, or maintenance of Ser expression. Although fj is expressed in every tarsal segment, Ser expression is only affected by loss of fj in tarsal segment 2 (Buckles, 2001).

To begin to understand how Fj signaling affects such diverse processes as leg segmentation and growth, ommatidial rotation, and epithelial planar polarity, attempts were made to identify other genes with which Fj interacts. Some of the effects of Fj are likely to be due to its feedback onto the N signaling pathway, and this would presumably require a Fj signal transduction pathway. In addition, it is likely that Fj also functions independently of its regulation of Ser and Dl, since the loss-of-function and gain-of-function phenotypes of N ligands and Fj are not identical (Buckles, 2001).

The predicted molecular structure of Fj suggests that it is a type II transmembrane protein, with two potential signal peptidase cleavage sites near the end of the transmembrane domain whose use would be consistent with the size of the secreted product. However, not all Fj is secreted, since two integral membrane forms remain present in all preparations. While there are many examples of growth factors that have both transmembrane and secreted forms, all of these appear to undergo cleavage at or near the cell surface, including N and its ligand Dl. If signal peptidase is indeed responsible for Fj cleavage (and this seems likely, as Fj is cleaved even in an in vitro microsomal preparation), it is unusually inefficient. Alternatively, it is possible that, in vivo, Fj is instead cleaved by a protease later in the secretory pathway or in response to signaling. This will need to be resolved in future experiments (Buckles, 2001).

It still remains to be determined which forms of Fj have functional significance. Certainly, the non-autonomous effects of Fj in the wing, eye and abdomen, all implicate secreted Fj as biologically relevant. Indeed, in vertebrates Fj appears to be produced as a wholly secreted protein with the transmembrane domain serving as an N-terminal signal sequence. In the gain-of-function clones described here, the induction of outgrowths composed of wild-type tissue similarly supports a non-autonomous role for Fj in the leg, although the non-autonomous influence may be indirect, for example by the early induction of a second growth factor. The failure of joint formation by wild-type tissue adjacent to these clones may also provide examples of non-autonomy. Alternatively, however, it is possible that an inhibition of joint formation within the clone mechanically inhibits nearby cells from forming joints. A similar local inhibitory influence has been observed on heterozygous cells adjacent to loss-of-function fj clones. Interestingly, the opposite is not observed: wild-type tissue is never seen to rescue joint formation within mutant clones. Again, this may represent the competing influences of induction from outside the clone and mechanical inhibition from within the clone. Thus the cooperative nature of joint development makes any determination of local non-autonomy ambiguous (Buckles, 2001).

Local suppression of joint formation adjacent to clones may also explain why ectopic joints are not seen along the borders of the flip-out clones, which produce sharp boundaries of expression that might be expected to resemble the normal patterning of fj expression. By contrast, ectopic joints are produced when fj is uniformly elevated in a wild-type background. While the significance of this remains unclear, it is noted that these ectopic structures tend to form in the center of the segment, where endogenous activity (and thus presumably additive activity) is lowest. Thus, in addition to the patterning of fj expression, the absolute level of Fj may determine whether joint initiation is permissible in any region of the disc (Buckles, 2001).

Similar mutant phenotypes may indicate that the genes causing them may act in the same molecular pathway. dachs and abl mutant phenotypes imitate those of fj, thus both of these genes are attractive candidates for the fj signaling pathway. A major substrate for Abl kinase activity is the Ena gene product. Ena homozygotes are embryonic lethal and imaginal phenotypes are not known. However, Abl and Ena appear to function in the same pathway in Drosophila. Finally, given the molecular epistatic interactions observed between fj and Ser, whether fj and Ser interact genetically was tested (Buckles, 2001).

To test the relationships of these genes, two hypomorphic alleles of fj, fj4 and fjN7 were used. The majority of legs of fjN7 flies retain partial joints of a ball and socket morphology at the juncture between T2 and T3, while fj4 produces larger partial joints or complete joints at the T2/3 boundary. Introduction of one mutant copy of dachs, abl, ena, or Ser into these backgrounds significantly increases the severity of the hypomorphic phenotypes, while each of these genes by itself is wholly recessive in the leg. Thus, dachs, abl, ena, and Ser act as dominant enhancers of fj, suggesting these genes may be part of a common pathway or network (Buckles, 2001).

Loss-of-function abl alleles are recessive, and heterozygous abl flies have normal leg morphology. However, when one copy of abl was removed in a strong fj background, the complete or partial loss of an additional tarsal joint at T1/2 on one or more legs was observed in one third of the animals. A wild-type abl transgene (Tnabl+) can restore this joint, confirming that abl is indeed responsible for the interaction. To test if kinase activity is required for abl activity at this joint, a transgene with an inactive kinase (TnablK-N) was inserted into the same genetic background. This transgene is unable to rescue the interaction, suggesting that abl kinase activity is indeed required. These results suggest that abl and fj participate in redundant pathways in the leg. Moreover, they demonstrate that fj is required at segment boundaries other than T2/3, which is the only boundary lost in fj null mutants. More widespread activity is consistent with the expression of fj at additional segment boundaries in the leg. These results reveal a concealed function for abl in leg morphogenesis (Buckles, 2001).

Abl kinase function partially masks a requirement for fj at the T1/2 segment boundary. While fj is expressed at all tarsal segment boundaries, it appears to be required for segmentation only at T2/3, although rare instances of partial loss of T1/2 have been observed. However, in the absence of one copy of abl, additional loss of the T1/2 boundary is seen in a third of all animals. This is interpreted to mean that additional information, modified by or working through abl, acts together with fj to elaborate that boundary. Most abl homozygous flies have normal legs, although occasional severe truncations of the legs in individual abl flies are observed. In addition, over-expression of abl causes tarsal truncations and segment fusions. A likely target of Abl activity is Ena. However, Abl is not the only tyrosine kinase that phosphorylates Ena, and this multiple regulation may in part explain the variability of abl loss-of-function phenotypes. A critical test of whether Ena is indeed part of a common pathway for the morphogenetic work of segmentation at all leg segment boundaries will be to examine loss-of-function ena clones in the leg. Alternatively, the common pathway at each segment boundary may be the actin cytoskeleton itself, with multiple inputs providing redundancy, and overlapping position-specific regulators competing or cooperating to regulate the state of actin dynamics in each cell. Indeed, in the larger sense, Fj itself may serve to coordinate information provided by multiple signaling pathways (Notch, Jak/Stat, and Wingless, which all regulate fj) with alterations in the actin cytoskeleton that ultimately have morphological consequences (Buckles, 2001).

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 wgspd-fg. At the same time, clones of cells mutant for fat overgrow in other imaginal cells, and fat wgspd-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).

Dachs: an unconventional myosin that functions downstream of Fat to regulate growth, affinity and gene expression in Drosophila

The dachs gene was first identified almost a century ago based on its requirements for appendage growth. This paper describes the phenotypes of strong dachs mutations, reports the cloning of the dachs gene, characterizes the localization of Dachs protein, and investigates the relationship between Dachs and the Fat pathway. Mutation of dachs reduces, but does not abolish, the growth of legs and wings. dachs encodes an unconventional myosin that preferentially localizes to the membrane of imaginal disc cells. dachs mutations suppress the effects of fat mutations on gene expression, cell affinity and growth in Imaginal discs. Dachs protein localization is influenced by Fat, Four-jointed and Dachsous, consistent with its genetic placement downstream of fat. However, dachs mutations have only mild tissue polarity phenotypes, and only partially suppress the tissue polarity defects of fat mutants. These results implicate Dachs as a crucial downstream component of a Fat signaling pathway that influences growth, affinity and gene expression during development (Mao, 2006).

The observation that a hypomorphic mutation of dachs could suppress the effects of fat mutations on wing growth and Wg expression in the proximal wing has led to the suggestion that dachs might act as a downstream component of a Fat signaling pathway. This study provides two types of evidence that confirm this suggestion. First, dachs is epistatic to fat for multiple phenotypes in multiple tissues, including gene expression, growth and cell affinity. Indeed, with the notable exception of the influence of fat on tissue polarity, all known fat mutant phenotypes are completely suppressed by mutation of dachs. Second, it was found that expression of regulators of Fat, Fj and Ds, or of Fat itself, influence the localization or stability of Dachs protein at the membrane, thus providing a molecular link from Fat to Dachs (Mao, 2006).

The predicted structure of Dachs is unique within the myosin superfamily, and places Dachs in a new class of unconventional myosins. It has most similarity to myosins V, VII, and X. This is intriguing, as a mammalian protocadherin, Cdh23, has been functionally linked to myosin VIIa during the development of sensory hair cells in the inner ear (Mao, 2006).

Within the myosin head region, the major conserved domains are all present, suggesting that Dachs functions as a motor protein. However, it is also possible that Dachs serves a structural or scaffolding role. For example, in the Hedgehog pathway, a kinesin-related protein, Costal2, is thought to function largely as a scaffold that brings together crucial kinases with their substrates (Mao, 2006).

The dGC2 mutation deletes part of the N terminal extension. As dGC2 mutants have relatively weak phenotypes, the N terminal extension might not be not essential for Dachs activity. Conversely, the severe phenotypes of alleles that truncate Dachs in the myosin head region imply that the myosin domain is essential. dGC13 in particular is predicted to eliminate almost all of the myosin head domain, and genetically it appears to act as a null allele (Mao, 2006).

Characterization of new dachs alleles has provided an opportunity to define more clearly the requirements for dachs. dachs is required for normal wing and leg growth, although some appendage growth is dachs independent. Importantly, the identification of dachs as a downstream component of a Fat signaling pathway that influences growth implies that the reduced growth in dachs mutants is reflective of a normal role for a Fat pathway in growth promotion. That is, while fat is a gene whose normal role can be thought of as to restrain growth, as mutant tissue overgrows, it is suggested that inhibition of Fat occurs during normal development, and that this inhibition contributes to normal appendage growth, as defined by the reduced growth of dachs mutants. Normal inhibition of Fat activity would presumably be effected by the two known regulators of Fat, Fj and Ds (Mao, 2006).

Whether available dachs mutations fully define the normal involvement of the Fat pathway in growth promotion is not yet clear. The possibility cannot be excluded that dachs is partially redundant with other proteins (e.g. other myosins), although this seems unlikely given the complete suppression of all non-polarity phenotypes of fat by dachs. It is also possible that dachs is required only for peak Fat signaling. This explanation is suggested by the observation that expression of the Fat target genes wg, Ser and fj is only partially or transiently lost in dachs mutants, yet the elevated or ectopic expression of these genes in fat mutants is completely eliminated by mutation of dachs (Mao, 2006).

The relatively mild tissue polarity phenotypes of dachs mutants, and the inability of dachs mutation to completely suppress the influence of fat on tissue polarity, contrast with the absolute dependence of fat gene expression, growth and affinity phenotypes on dachs. These observations suggest that there are two distinct Fat pathways. One, crucially dependent on Dachs, influences gene expression, growth and cell affinity, and another, partially independent of Dachs, influences tissue polarity. Studies of the atrophin protein Grunge also support the suggestion that there is a distinct Fat polarity pathway, as Grunge interacts with Fat and influences tissue polarity, but does not exhibit other phenotypes observed in fat mutants. Thus, Dachs might act redundantly with another protein in a polarity pathway, but non-redundantly in a pathway that influences gene expression. It should also be noted that effects of dachs on gene expression might contribute to the polarity phenotypes of dachs mutants. For example, fj is regulated by dachs, and fj has polarity phenotypes (Mao, 2006).

The asymmetric localization of Dachs observed in wild-type wings, and the influence of Fj and Ds on Dachs localization, have important implications for tissue polarity. (1) The asymmetric localization of Dachs is itself a form of polarity, and its detection in third instar imaginal discs emphasizes that these cells are polarized well before core polarity proteins such as Frizzled and Dishevelled become asymmetrically localization in pupal wings. A similar conclusion can be drawn from the recent observation that fat and ds influence the orientation of cell divisions in third instar discs. (2) The observations identify an ability to induce asymmetric protein localization as a mechanism through which the Fat pathway might influence tissue polarity. Dachs is one target, but the Fat polarity pathway might similarly involve asymmetric localization of other myosins, or of other types of proteins, to affect tissue polarity (Mao, 2006).

Mutation of fat is associated with elevated Dachs staining at the membrane, and overexpression of Fat decreases Dachs staining at the membrane. Although this negative effect of Fat on Dachs is subject to the caveat that only tagged overexpressed Dachs:V5 can be detected, this tagged protein rescues dachs mutants, and the effects of Fat on Dachs staining are consistent with their opposite phenotypes and the epistasis of dachs to fat. Manipulations of the expression of Fat regulators provide further evidence that Fat regulates Dachs levels at the membrane, and altogether these observations implicate Dachs as a crucial intracellular component of a Fat signaling pathway (Mao, 2006).

The concomitant elevation of Fat staining and loss of Dachs staining observed at the perimeter of Fj-expressing clones is consistent with the conclusion that Fat can antagonize the localization or stability of Dachs at the membrane. Because the elevation of Fat is limited to the periphery of Fj-expressing clones, it is hypothesized that it results from an influence of Fj on Fat-Ds interactions, rather than the expression of Fj per se. Tissue polarity studies have implied that Fj and Ds have opposite affects on Fat. Although it has not yet been determined whether Fj can directly modify Fat or Ds, the simplest explanation for the elevated Fat staining at the edge of Fj-expressing cells would be to propose that Fj modifies Ds to inhibit its interactions with Fat. In this case, Fat protein within Fj-expressing clones would be predicted to prefer to bind to Ds outside of the clone, and hence to accumulate at the clone perimeter, where it would then downregulate Dachs (Mao, 2006).

The interpretation of the elevated Dachs staining at the perimeter of Ds-expressing clones is more complex. Although Fat is elevated at the clone perimeter, the depletion of Fat from neighboring cells suggests that the elevated Fat staining largely reflects Fat outside of the clone, rather than in Ds-expressing cells. Given that dachs and fat influence transcriptional targets cell autonomously, and dachs acts genetically downstream of fat, the link between elevated Fat in one cell and elevated Dachs in a neighboring cells must be indirect. It might be that Ds can also influence Dachs localization, and does so in opposite fashion to Fat. According to this scenario, the elevated Fat staining in cells neighboring the clone would be reflective of high levels of Ds engaged by Fat at the clone perimeter, which would then recruit or stabilize Dachs at the membrane. However, mutation of ds did not result in any noticeable decrease of Dachs:V5 staining. Alternatively, it might be that Fat antagonizes the accumulation of Ds within the same cell. High Fat accumulation at the edge of one cell could then result in low Fat accumulation at the edge of its neighbor through this hypothesized downregulation of Ds. In this case, the elevated Dachs accumulation at the edge of Ds-expressing clones would be a consequence of low levels of Fat. This model would also imply that asymmetric localization of Fat could be propagated from cell to cell, which could have important consequences for Fat pathway regulation. However, there is as yet no evidence that Fat is asymmetrically localized at wild-type levels of Fj and Ds expression (Mao, 2006).

Delineation of a Fat tumor suppressor pathway

Recent studies in Drosophila of the protocadherins Dachsous and Fat suggest that they act as ligand and receptor, respectively, for an intercellular signaling pathway that influences tissue polarity, growth and gene expression, but the basis for signaling downstream of Fat has remained unclear. This study characterizes functional relationships among Drosophila tumor suppressors and identifies the kinases Discs overgrown and Warts as components of a Fat signaling pathway. fat, discs overgrown and warts regulate a common set of downstream genes in multiple tissues. Genetic experiments position the action of discs overgrown (dco) upstream of the Fat pathway component dachs, whereas warts acts downstream of dachs. Warts protein coprecipitates with Dachs, and Warts protein levels are influenced by fat, dachs and discs overgrown in vivo, consistent with its placement as a downstream component of the pathway. The tumor suppressors Merlin, expanded (ex), hippo, salvador (sav) and mob as tumor suppressor (mats) also share multiple Fat pathway phenotypes but regulate Warts activity independently. These results functionally link what had been four disparate groups of Drosophila tumor suppressors, establish a basic framework for Fat signaling from receptor to transcription factor and implicate Warts as an integrator of multiple growth control signals (Cho, 2006).

Since Dachs is required for loss of Wts protein in fat mutants, and Dachs encodes a large Myosin protein, a model was considered in which Dachs acts as a scaffold to link Wts to proteins that promote Wts proteolysis, analogous to the roles of Costal2 in Hedgehog signaling, or APC in Wnt signaling. This model predicts that Dachs should be able to bind to Wts. To evaluate this possibility, tagged forms of Dachs and Wts were coexpressed in cultured cells and assayed for coimmunoprecipitation. These experiments identified a specific and reproducible interaction between Dachs and Wts (Cho, 2006).

Recent studies have identified the transcriptional coactivator Yorkie (Yki) as a downstream component of the Hippo pathway and a substrate of Wts kinase activity. Phosphorylation of Yki by Wts inactivates Yki, and overexpression of Yki phenocopies wts mutation. The determination that the Fat tumor suppressor pathway acts through modulation of Wts thus predicts that Yki should also be involved in Fat signaling. When the influence of Yki overexpression was examined on Fat target genes, expression of Wg in the proximal wing, Ser in the proximal leg and fj in the wing and eye were each upregulated by Yki overexpression, consistent with the inference that Fat tumor suppressor pathway signaling acts through Yki (Cho, 2006).

In order to identify additional components of the Fat tumor suppressor pathway, advantage was taken of the observation that loss of fat in clones of cells is associated with an induction of Wingless (Wg) expression in cells just proximal to the normal ring of Wg expression in the proximal wing, reflective of its role in distal-to-proximal wing signaling. It was reasoned that this influence on Wg expression could be used to screen other Drosophila tumor suppressors for their potential to contribute to Fat signaling. Analysis of mutant clones in the proximal wing identified dco, ex, mats, sav, hpo and wts as candidate components of the Fat tumor suppressor pathway. As for fat, mutation of each of these genes is associated with induction of Wg expression specifically in the proximal wing, whereas Wg expression is not affected in more distal or more proximal wing cells. Although Wg expression often seems slightly elevated within its normal domain, the effect of these mutations is most obvious in the broadening of the Wg expression ring. The induction of Wg expression does not seem to be a nonspecific consequence of the altered growth or cell affinity associated with these mutations, since Wg expression is unaffected by expression of the growth-promoting microRNA gene bantam or by expression of genes that alter cell affinity in the proximal wing (Cho, 2006).

dco encodes D. melanogaster casein kinase I delta/epsilon. The overgrowth phenotype that gave the gene its name is observed in allelic combinations that include a hypomorphic allele, dco3, and it is this allele that is associated with induction of Wg. Null mutations of dco actually result in an 'opposite' phenotype: discs fail to grow, and clones of cells mutant for null alleles fail to proliferate. This is likely to reflect requirements for dco in multiple, distinct processes, as casein kinase I proteins phosphorylate many different substrates, and dco has been implicated in circadian rhythms, Wnt signaling and Hedgehog signaling (Cho, 2006).

Mer and ex encode two structurally related FERM domain-containing proteins. ex was first identified as a Drosophila tumor suppressor, whereas Drosophila Mer was first identified based on its structural similarity to human Merlin. Mutation of Mer alone causes only mild effects on imaginal disc growth, but Mer and ex are partially redundant, and double mutants show more severe overgrowth phenotypes than either single mutant. Consistent with this, elevation of Wg expression was observed in ex mutant clones (7/10 proximal wing clones induced Wg) and not in Mer mutant clones (0/8 clones), whereas Mer ex double mutant clones showed even more severe effects on Wg than ex single mutant clones. Because of the partial redundancy between Mer and ex, when possible, focus was placed for subsequent analysis on Mer ex double mutant clones (Cho, 2006).

Wts, Mats, Sav and Hpo interact biochemically, show similar overgrowth phenotypes and regulate common target genes. Mats, Sav and Hpo are all thought to act by regulating the phosphorylation state and thereby the activity of Wts. Mutation of any one of these genes is associated with upregulation of Wg in the proximal wing. The effects of sav (47/84 clones in the proximal wing induced Wg) and hpo (23/31 clones) were weaker than those of mats (19/19 clones) and wts (92/97 clones), but this might result from differences in perdurance or allele strength. Because sav, hpo and mats all act through Wts, focus for most of the subsequent analysis was placed on wts (Cho, 2006).

The observation that mutation of dco, Mer, ex, mats, sav, hpo or wts all share the distinctive upregulation of Wg expression in the proximal wing observed in fat mutants suggests that the functions of these genes are closely linked. To further investigate this, the effects of these tumor suppressors were characterized on other transcriptional targets of Fat signaling. Expression of the Notch ligand Ser is upregulated unevenly within fat mutant cells in the proximal region of the leg disc. A very similar upregulation occurred in dco3, Mer ex, and wts mutant clones. fj is a target of Fat signaling in both wing and eye imaginal discs, and fj expression was also upregulated in dco3, Mer ex, or wts mutant clones. The observation that these genes share multiple transcriptional targets in different Drosophila tissues implies that they act together in a common process (Cho, 2006).

The hypothesis that Fat pathway genes and Hippo pathway genes are linked predicts that not only should Fat target genes be regulated by Hippo pathway genes, but Hippo pathway target genes should also be regulated by Fat pathway genes. The cell cycle regulator CycE and the inhibitor of apoptosis Diap1 (encoded by thread) have been widely used as diagnostic downstream targets to assign genes to the Hippo pathway. Notably, then, clones of cells mutant for fat showed upregulation of both Diap1 and CycE protein expression. Genes whose expression is upregulated within fat mutant cells (such as wg, Ser and fj) have been shown previously to be induced along the borders of cells expressing either fj or dachsous (ds), and Diap1 is also upregulated around the borders of ds- or fj-expressing clones. That thread is affected by fat at a transcriptional level was confirmed by examining a thread-lacZ enhancer trap line. The regulation of Diap1 by the Hippo pathway is thought to be responsible for a characteristic eye phenotype in which an excess of interommatidial cells results from their failure to undergo apoptosis; an increase was also observed in interommatidial cells in fat mutant clones. Upregulation of both Diap1 and CycE is also observed in Mer ex double mutant clones. In dco3 mutant clones, consistent upregulation was detected only for Diap1, and CycE was upregulated only weakly and inconsistently. dco3 also has weaker effects on Wg and fj expression; the weaker effects of dco3 could result from its hypomorphic nature. ex has recently been characterized as another Hippo pathway target, and an ex-lacZ enhancer trap that is upregulated in wts or Mer ex mutant clones is also upregulated in fat or dco3 mutant clones. Analysis of ex transcription by in situ hybridization also indicated that ex is regulated by fat. Altogether, this analysis of Hippo pathway targets further supports the conclusion that the functions of the Fat pathway, the Hippo pathway and the tumor suppressors Mer, ex and dco are linked (Cho, 2006).

Genetic epistasis experiments provide a critical framework for evaluating the functional relationships among genes that act in a common pathway. The relationships was evaluated between each of the tumor suppressors linked to the Fat pathway and dachs, using both wing disc growth and proximal Wg expression as phenotypic assays. dachs is the only previously identified downstream component of the Fat tumor suppressor pathway. It acts oppositely to fat and is epistatic to fat in terms of both growth and gene expression phenotypes (Cho, 2006).

dachs is also epistatic to dco3 for overall wing disc growth and for proximal Wg expression. The epistasis of dachs to dco3 implies that the overgrowth phenotype of dco3 is specifically related to its influence on Fat signaling, as opposed to participation of dco in other pathways. By contrast to the epistasis of dachs to dco3, both wts and ex are epistatic to dachs for disc overgrowth phenotypes, and wts and Mer ex are epistatic to dachs in their influence on proximal Wg expression. Together, these epistasis experiments suggest that dco acts upstream of dachs, whereas Mer ex and wts act downstream of dachs (Cho, 2006).

Because wts and Mer ex have similar phenotypes, their epistatic relationship cannot be determined using loss-of-function alleles. However, overexpression of ex inhibits growth and promotes apoptosis, which suggests that ex overexpression affects ex gain-of-function. Clones of cells overexpressing ex are normally composed of only a few cells, and over time most are lost, but coexpression with the baculovirus apoptosis inhibitor p35 enabled recovery of ex-expressing clones. These ex- and p35-expressing clones were associated with repression of proximal Wg expression during early- to mid-third instar, as has been described for dachs2, consistent with ex overexpression acting as a gain-of-function allele in terms of its influence on Fat signaling. In epistasis experiments using overexpressed ex and mutation of wts, wts was epistatic; Wg was induced in the proximal wing. Additionally, when wts is mutant, coexpression with p35 was no longer needed to ensure the viability and growth of ex-expressing clones, indicating that wts is also epistatic to ex for growth and survival. Consistent with this conclusion, others have recently described phenotypic similarities between Mer ex and hpo pathway mutants and have reported that hpo is epistatic to Mer ex (Cho, 2006).

When Fat was overexpressed, a slight reduction was detected in Wg expression during early- to mid-third instar, suggesting that overexpression can result in a weak gain-of-function phenotype. Clones of cells overexpressing Fat but mutant for dco3 still showed reduced Wg levels, whereas clones of cells overexpressing Fat but mutant for warts showed increased Wg levels. Although experiments in which the epistatic mutation is not a null allele cannot be regarded as definitive, these results are consistent with the conclusion that wts acts downstream of fat and suggest that dco might act upstream of fat (Cho, 2006).

The epistasis results described above suggest an order of action for Fat tumor suppressor pathway genes in which dco acts upstream of fat, fat acts upstream of dachs, dachs acts upstream of Mer and ex, and Mer and ex act upstream of wts. However, the determination that one gene is epistatic to another does not prove that the epistatic gene is biochemically downstream, as it is also possible that they act in parallel but converge upon a common target. Thus, to better define the functional and hierarchical relationships among these genes, experiments were initiated to investigate the possibility that genetically upstream components influence the phosphorylation, stability or localization of genetically downstream (that is, epistatic) components. Focus in this study was placed on the most downstream of these components, Wts. As available antibodies did not specifically recognize Wts in imaginal discs, advantage was taken of the existence of functional, Myc-tagged Wts-expressing transgenes (Myc:Wts) to investigate potential influences of upstream Fat pathway genes on Wts protein. In wing imaginal discs, Myc:Wts staining outlines cells, suggesting that it is preferentially localized near the plasma membrane, and it was confirmed that expression of Myc:Wts under tub-Gal4 control can rescue wts mutation. Notably, mutation of fat results in a reduction of Myc:Wts staining. As Myc:Wts is expressed under the control of a heterologous promoter in these experiments, this must reflect a post-transcriptional influence on Wts protein. fat does not exert a general influence on the levels of Hippo pathway components; fat mutant clones had no detectable influence on the expression of hemagglutinin epitope-tagged Sav (HA:Sav) (Cho, 2006).

The decrease in Wts protein associated with mutation of fat contrasts with studies of the regulation of Wts activity by the Hippo pathway, which have identified changes in Wts activity due to changes in its phosphorylation state. To directly compare regulation of Wts by Fat with regulation of Wts by other upstream genes, Myc:Wts staining was examined in ex, sav and mats mutant clones. In each of these experiments, the levels and localization of Myc:Wts in mutant cells was indistinguishable from that in neighboring wild-type cells (Cho, 2006).

Since Myc:Wts appears preferentially localized near the plasma membrane, it was conceivable that the apparent decrease in staining reflected delocalization of Wts, rather than destabilization. To investigate this possibility, Wts levels were examined by protein blotting. Antisera against endogenous Wts recognized a band of the expected mobility in lysates of wing imaginal discs or cultured cells, and this band was enhanced when Wts was overexpressed. The intensity of this band was reproducibly diminished in fat or dco3 homozygous mutant animals but was not diminished in fat or dco3 heterozygotes or in ex mutants. Conversely, levels of Hpo, Sav, Mer or Mats were not noticeably affected by fat mutation (Cho, 2006).

The determination that Wts is affected by Fat, together with the genetic studies described above, place Wts within the Fat signaling pathway, as opposed to a parallel pathway that converges on common transcriptional targets. Indeed, given that even hypomorphic alleles of wts result in disc overgrowth, the evident reduction in Wts levels might suffice to explain the overgrowth of fat mutants. As a further test of this possibility, Wts levels were examined in fat dachs double mutants. As the influence of Fat on gene expression and growth is absolutely dependent upon Dachs, if Fat influences growth through modulation of Wts, its influence on Wts levels should be reversed by mutation of dachs. Examination of Myc:Wts staining in fat dachs clones and of Wts protein levels in fat dachs mutant discs confirmed this prediction (Cho, 2006).

Prior observations, including the influences of fat and ds on gene expression, and the ability of the Fat intracellular domain to rescue fat phenotypes, suggested that Fat functions as a signal-transducing receptor. By identifying kinases that act both upstream (Dco) and downstream (Wts) of the Fat effector Dachs and by linking Fat to the transcriptional coactivator Yki, these results have provided additional support for the conclusion that Fat functions as a component of a signaling pathway and have delineated core elements of this pathway from receptor to transcription factor. Fat activity is regulated, in ways yet to be defined, by Ds and Fj. The influences of Fat on gene expression, growth, and cell affinity, as well as on Wts stability, are completely dependent on Dachs, indicating that Dachs is a critical effector of Fat signaling. Since Dachs can associate with Wts or a Wts-containing complex, it is suggested that Dachs might act as a scaffold to assemble a Wts degradation complex. The observations that Fat, Ds and Fj modulate the subcellular localization of Dachs, that Wts is preferentially localized near the membrane and that Dachs accumulates at the membrane in the absence of Fat, suggest a simple model whereby Fat signaling regulates Wts stability by modulating the accumulation of Dachs at the membrane and thereby its access to Wts. The working model is that dco3 is defective in the phosphorylation of a substrate in the Fat pathway, but the recessive nature of dco3, the genetic epistasis experiments, and biochemical experiments argue that this substrate is not Wts, and further work is required to define the biochemical role of Dco in Fat signaling (Cho, 2006).

In addition to identifying core components of the Fat pathway, the results establish close functional links between the Fat pathway, the Hippo pathway and the FERM-domain tumor suppressors Mer and Ex. The common phenotypes observed among these tumor suppressors can be explained by their common ability to influence Wts. However, they seem to do this in distinct ways, acting in parallel pathways that converge on Wts rather than a single signal transduction pathway. The Fat pathway modulates levels of Wts, apparently by influencing Wts stability. By contrast, the Hippo pathway seems to regulate the activity of Wts by modulating its phosphorylation state. Thus, Wts seems to act as an integrator of distinct growth signals, which can be transmitted by both the Fat pathway and the Hippo pathway. It has been suggested that Mer and Ex also act through the Hippo pathway, although present experiments cannot exclude the possibility that Mer and Ex act in parallel to Hpo. Moreover, it should be noted that Mats might regulate Wts independently of Hpo and Sav and hence function within a distinct, parallel pathway. Although it is simplest to think of parallel pathways, there is also evidence for cross-talk. fj and ex are both components and targets of these pathways. Thus, they can be regarded as feedback targets within their respective pathways, but their regulation also constitutes a point of cross-talk between pathways. Another possible point of cross-talk is suggested by the observation that levels of Fat are elevated within Mer ex mutant clones. Although the potential for cross-talk complicates assessments of the relationships between tumor suppressors, the observations that fat, dco3 and dachs affect Warts protein levels in vivo, whereas ex, hippo, sav and mats do not, argues that there are at least two distinct pathways that converge on Warts. This conclusion is also consistent with the observations that ex, hippo, sav and mats can influence Wts phosphorylation in cultured cell assays, but Fat, Dachs and Dco do not (Cho, 2006).

Although the Fat and Hippo pathways converge on Wts, Hippo pathway mutants seem more severe. Thus, hpo, wts or mats mutant clones show a distinctive disorganization and outgrowth of epithelial tissues that is not observed in fat mutant clones, and they show a greater increase in interommatidial cells. This difference presumably accounts for the previous failure to recognize the tight functional link between Fat and Hippo signaling, and it can be explained by the finding that Wts levels are reduced but not completely absent in fat mutant cells. Thus, fat would be expected to resemble a hypomorphic allele of wts rather than a null allele, and consistent with this, a hypomorphic allele, wtsP2, results in strong overgrowth phenotypes. The effects of Yki overexpression on growth and target gene expression can be even stronger than those of fat or wts mutations, which suggests that Yki levels become limiting when upstream tumor suppressors are mutant (Cho, 2006).

fat encodes a protocadherin, which in the past has led to speculation that its influences on growth and cell affinity might result from Fat acting as a cell adhesion molecule. However, all of the effects of fat on growth and affinity require dachs, which is also required for the effects of fat on transcription. Additionally, targets of Fat signaling include genes that can influence growth and affinity; recent studies identified an influence of fat on E-cadherin expression, and as describe in this study, Fat influences CycE and Diap1 expression. Thus, one can account for the influence of fat on growth and affinity by its ability to regulate gene expression. fat interacts genetically with other signaling pathways, including EGFR and Wnt, and in some cells Fat signaling also influences the expression of ligands (such as Wg and Ser) for other signaling pathways. Regulation of these ligands contributes to fat overgrowth phenotypes, but since clonal analysis indicates that fat is autonomously required for growth control in most imaginal cells, the principal mechanism by which fat influences growth presumably involves the regulation of general targets (Cho, 2006).

Normal tissue growth and patterning depend on a relatively small number of highly conserved intercellular signaling pathways. The Fat pathway is essential for the normal regulation of growth and PCP in most or all of the external tissues of the fly and also participates in local cell fate decisions. In this regard, its importance to fly development can be considered comparable to that of other major signaling pathways. Although the biological roles and even the existence of a Fat pathway in mammals remain to be demonstrated, there is clear evidence that the mammalian Warts homologs Lats1 and Lats2 act as tumor suppressors and that a mammalian Yorkie homolog, YAP, can act as an oncogene. Moreover, other genes in the Drosophila Fat pathway have apparent structural homologs in mammals. Thus, it is likely that mammals also have a Fat tumor suppressor pathway that functions in growth control (Cho, 2006).

Morphogen control of wing growth through the fat signaling pathway

Organ growth is influenced by organ patterning, but the molecular mechanisms that link patterning to growth have remained unclear. The Dpp morphogen gradient in the Drosophila wing influences growth by modulating the activity of the Fat signaling pathway. Dpp signaling regulates the expression and localization of Fat pathway components, and Fat signaling through Dachs is required for the effect of the Dpp gradient on cell proliferation. Juxtaposition of cells that express different levels of the Fat pathway regulators four-jointed and dachsous stimulates expression of Fat/Hippo pathway target genes and cell proliferation, consistent with the hypothesis that the graded expression of these genes contributes to wing growth. Moreover, uniform expression of four-jointed and dachsous in the wing inhibits cell proliferation. These observations identify Fat as a signaling pathway that links the morphogen-mediated establishment of gradients of positional values across developing organs to the regulation of organ growth (Rogulja, 2008).

Studies of regeneration first led to models that proposed that growth could be influenced by gradients of positional values, with steep gradients promoting growth and shallow gradients suppressing growth. Experimental manipulations of Dpp pathway activity in the Drosophila wing supported this concept, but have left unanswered the question of how differences in the levels of Dpp pathway activity perceived by neighboring cells are actually linked to growth. This study has established that the Fat signaling pathway provides this link. Dpp signaling influences the Fat pathway; the expression of upstream Fat pathway regulators, the subcellular localization of Fat pathway components, and downstream transcriptional outputs of Fat signaling are all affected by Dpp signaling. The effects that Tkv and Brk expression have on the expression of Fat target genes parallels their effects on BrdU labeling and depend genetically on Fat signaling (Rogulja, 2008).

Dpp signaling impinges on Fat signaling upstream of Fat, as the expression of both of its known regulators, Fj and Ds, is regulated by Dpp signaling. Although the Fat signaling pathway was only recently discovered, and understanding of Fat signaling and its regulation remains incomplete, the inference that Fat signaling is normally influenced by the Dpp morphogen gradient is supported by the polarized localization of Dachs in wild-type wing discs. Near the D-V compartment boundary, the vector of Dachs polarization parallels the vector of the Dpp morphogen gradient, and the consequences of altered Dpp pathway activity confirm that the correlation between them is reflective of a functional link. The expression of Fj and Ds and the localization of Dachs are also polarized along the D-V axis. The implication that signaling downstream of the D-V compartment boundary thus also impinges on Fat signaling, and indeed may also influence growth through this pathway, is consistent with the observation that normal wing growth requires both A-P and D-V compartment boundary signals, and is further supported here by the observation that Notch activation affects both fj expression and Dachs localization (Rogulja, 2008).

The results argue that Fat signaling is influenced by the graded expression of its regulators: uniform expression of Fj and Ds can activate Fat signaling and thereby inhibit growth, whereas juxtaposition of cells expressing different levels of either Fj or Ds can inhibit Fat signaling and thereby promote growth. Here, a model is proposed to explain how Fat signaling can be modulated by Fj and Ds gradients. Although aspects of the model remain speculative, it provides an explanation for a number of observations that would otherwise appear puzzling, and serves as a useful framework for future studies (Rogulja, 2008).

Central to the model is the inference that the interaction between Ds and Fat activates Fat. This inference is well supported by the observations that mutation or downregulation of ds results in overgrowth and upregulation of Diap1, whereas uniform overexpression of Ds inhibits growth and Diap1 expression. A second key aspect of the model is that once activated by Ds, Fat locally transmits a signal to a complex at the membrane. An important corollary to this is that if Fat and Ds are not engaged around the entire circumference of a cell, then there could be a region where Fat is locally inactive. This is hypothetical, but the Fat-dependent polarization of Dachs implies that there can be regional differences in Fat activity within a cell. Local Fat signaling is then proposed to locally promote Warts stability and activity, and thereby locally antagonize Yki activity. Conversely, a local absence of Fat signaling could result in a local failure to phosphorylate Yki, which could then transit to the nucleus, where it would promote the expression of downstream target genes. Formally, this model treats Fat signaling like a contact inhibition pathway: if Fat is engaged by Ds around the entire circumference of a cell, then Fat is active everywhere and downstream gene expression is off; however, if Fat is not active on even one side of a cell, then Yki-dependent gene expression can be turned on and growth can be promoted (Rogulja, 2008).

In this model, graded expression of Fat regulators, like Fj and Ds, could modulate Fat signaling by polarizing Fat activity within a cell. In theoretical models of PCP, even shallow gradients of polarizing activity can be converted to strong polarity responses through positive-feedback mechanisms. How this might be achieved in Fat signaling is not yet clear, but the polarized localization of Dachs implies that, at some level, Fat activity is normally polarized in wild-type animals, even where the Fj and Ds expression gradients appear relatively shallow. Importantly, this polarization hypothesis provides a solution to the puzzle of how Ds could act as a ligand to activate Fat, yet inhibit Fat along the edges of Ds-expressing clones. In this model, Ds overexpression in clones polarizes Fat activity, possibly through its ability to relocalize Fat. This would allow a strong derepression of Yki on the side of the cell opposite to where Ds and Fat are actually bound, resulting in the induction of Yki:Scalloped target gene expression and promotion of cell proliferation. Propagation of this polarization, e.g., through the influence of Fat-Ds binding on Fat and Ds localization, might explain the spread of effects beyond immediately neighboring cells. Conversely, uniform expression of Ds would generate cells presenting a ligand that activates Fat and dampens the relative difference in expression levels between neighboring cells. Yki would thus remain sequestered around the entire cell circumference, consistent with the reduced growth and Diap1 expression observed. A dampening of gradients could also explain why the induction of Fat/Hippo target gene expression or BrdU labeling associated with clones expressing Ds, Fj, or TkvQ-D is biased toward cells outside of clones (Rogulja, 2008).

The hypothesis of Fat polarization and local signal transduction also suggests a solution to another puzzle. In terms of their effects on tissue polarity and Dachs localization, Fj and Ds always behave as though they have opposite effects on Fat. Conversely, in terms of their effects on cell proliferation and downstream gene expression, Fj and Ds behave as though they have identical effects on Fat. To explain this, it is proposed that Fj acts oppositely to Ds, by, for example, antagonizing Ds-Fat binding. The influence of Ds and Fj on polarity would be a function of the direction in which they polarize Fat activity, which, based on their effects on epitope-tagged protein Dachs:V5, is opposite. In contrast, their influence on downstream gene expression and growth would be a function of the degree to which they polarize Fat activity, which could be the same. In other words, their influence on polarity would be a function of the vector of their expression gradients, and their influence on growth would be a function of the slope. However, since Dachs:V5 generally appears to be strongly polarized, the actual interpretation of Fj and Ds gradients may involve feedback amplification and threshold responses rather than providing a continuous response proportional to the gradient slope (Rogulja, 2008).

The results have provided a molecular understanding of a how a gradient of positional values, established by the morphogen Dpp and reflected, at least in part, in the graded expression of Fj and Ds, can influence growth. However, it is clear that other mechanisms must also contribute to the regulation of wing growth. The relative contribution of Fat gradients to wing growth can be estimated by considering the size of the wing in dachs mutants, or when Fj and Ds are expressed ubiquitously, as, in either case, it would be expected that the derepression of Yki associated with normal Fat signaling gradients was abolished. In both cases, the wing is less than half its normal size. Fat signaling could thus be considered a major, but by no means the sole, mechanism for regulating wing growth. The determination that not all wing growth depends on the regulation of Fat activity fits with the observation that Dpp signaling promotes growth in at least two distinct ways, one dependent upon its gradient, and the other dependent upon its levels. Other models for wing growth, including a Vestigial-dependent recruitment of new cells into the wing, and an inhibition of Dpp-promoted wing growth by mechanical strain, have also been proposed. It is emphasized that these models are not incompatible with the conclusion that a Fat gradient influences growth. Rather, it is plausible, and even likely, that multiple mechanisms contribute to the appropriate regulation of wing growth. Indeed, it is expected that a critical challenge for the future will be to define not only the respective contributions of these or other mechanisms to growth control, but also to understand feedback and crosstalk processes that influence how these different mechanisms interact with each other (Rogulja, 2008).

Propagation of Dachsous-Fat planar cell polarity

The Fat pathway controls both planar cell polarity (PCP) and organ growth. Fat signaling is regulated by the graded expression of the Fat ligand Dachsous (Ds) and the cadherin-domain kinase Four-jointed (Fj). The vectors of these gradients influence PCP, whereas their slope can influence growth. The Fj and Ds gradients direct the polarized membrane localization of the myosin Dachs, which is a crucial downstream component of Fat signaling. This study shows that repolarization of Dachs by differential expression of Fj or Ds can propagate through the wing disc, which indicates that Fj and Ds gradients can be measured over long range. Through characterization of tagged genomic constructs, it was shown that Ds and Fat are themselves partially polarized along the endogenous Fj and Ds gradients, providing a mechanism for propagation of PCP within the Fat pathway. A biochemical mechanism was identified that might contribute to this polarization by showing that Ds is subject to endoproteolytic cleavage and that the relative levels of Ds isoforms are modulated by Fat (Ambegaonkar, 2012).

The observation that differences in Fj or Ds expression can alter Fat PCP at a distance and that Ds, and to a lesser extent Fat, is polarized within the wing, together with other recent studies, imply that establishment of polarity in the Fat PCP system relies not just upon direct interpretation of Fj and Ds gradients but also upon amplification and propagation of PCP. To achieve this, PCP models incorporate both asymmetric intercellular signaling and antagonistic intracellular interactions between complexes that localize to distinct sides. Intercellular binding between Ds and Fat is well established, but on its own, this would not propagate polarity from cell to cell. However, incorporation of a local, intracellular antagonism of Ds by Fat activity could polarize Ds localization, which could then enable Fat-PCP to propagate. It is hypothesized that Fat regulates Ds by influencing production or stability of processed Ds isoforms (Ambegaonkar, 2012).

The propagation of polarity means that Fat-PCP is influenced not only by the local gradient but also by differential expression at a distance. Strong repolarization of Dachs was dependent upon having substantial differences in expression. Notably, strong differences in expression of both Fj and Ds normally occur in the proximal wing, and these differences have significant effects on Fat activity. Both measures of the range of Dachs repolarization and mathematical modeling suggest that the Fj/Ds expression boundary in the proximal wing would not be sufficient to direct Fat-PCP across 30 or more cells, as would be required at late third instar. However, at early third instar, when the developing wing is small, a mechanism that propagates PCP from an expression boundary for several cells could in principle be sufficient to establish PCP throughout the wing. Once established, the mechanisms that allow Fat-PCP to propagate could also help maintain Fat-PCP as the wing grows. In this case, the Fj and Ds boundaries at the edge of the developing wing would be the main drivers of polarity, rather than the shallow gradients of their expression within the wing itself (Ambegaonkar, 2012).

Integrating force-sensing and signaling pathways in a model for the regulation of wing imaginal disc size

The regulation of organ size constitutes a major unsolved question in developmental biology. The wing imaginal disc of Drosophila serves as a widely used model system to study this question. Several mechanisms have been proposed to have an impact on final size, but they are either contradicted by experimental data or they cannot explain a number of key experimental observations and may thus be missing crucial elements. This study has modeled a regulatory network that integrates the experimentally confirmed molecular interactions underlying other available models. Furthermore, the network includes hypothetical interactions between mechanical forces and specific growth regulators, leading to a size regulation mechanism that conceptually combines elements of existing models, and can be understood in terms of a compression gradient model. According to this model, compression increases in the center of the disc during growth. Growth stops once compression levels in the disc center reach a certain threshold and the compression gradient drops below a certain level in the rest of the disc. This model can account for growth termination as well as for the paradoxical observation that growth occurs uniformly in the presence of a growth factor gradient and non-uniformly in the presence of a uniform growth factor distribution. Furthermore, it can account for other experimental observations that argue either in favor or against other models. The model also makes specific predictions about the distribution of cell shape and size in the developing disc, which were confirmed experimentally (Aegerter-Wilmsen, 2012).

This paper presents a new model for the regulation of wing disc size. The model contains a rather complex regulatory network, which consists of a considerable number of interactions, receives nonuniform input of protein activities, and interacts with a mechanical stress pattern that emerges over time and space. It is assumed that the regulatory network represents protein activities and interactions that regulate these activities. The model does not distinguish between interactions at the transcriptional and protein activity level, but considers effects on net activities. All protein activities emerge from the network, except for those of Dpp, Wg and N, which are implemented in the model. In the regulatory network, differences in Ds and Fj concentrations between neighboring cells lead to activation of Dichate (D) by changing its intracellular localization. In addition, it is assumed that a weighted average of the area of a cell and its neighbors is a good readout for mechanical stress, that cells do not rearrange when exposed to mechanical tension, and that the planar polarization of D imposes a bias on the direction of the division plane. The interactions are hypothetical and form the main untested assumptions underlying the model. The regulation of ds by mechanical compression is not essential for the principle behind size regulation in the model, but improves the fit of simulation results with experimental data (Aegerter-Wilmsen, 2012).

A qualitative understanding can be gained by considering it in terms of a compression gradient model. During growth, compression increases in the center of the disc. Growth ceases when compression in the center reaches a certain threshold and the gradient of the compression gradient drops below a certain threshold in the rest of the disc. Read-out of the compression gradient is accomplished by a mechanism that involves Vg and the Hippo pathway. Numerical simulations were used to show that the model can account for growth termination and that it reproduces a large range of additional data on growth regulation, including some emergent properties of the system. Based upon the principle underlying the model, predictions can be made with respect to cell shape patterns. In order to take into account the curved surface of the wing pouch, an open source image analysis program was developed. The results showed that the general dynamics of the formation of cell shape patterns is indeed similar to the one predicted by the model. This analysis is, however, based on images from different discs and, especially during the early stages, there is variation among discs. It would therefore be interesting to assess whether the predicted dynamics is also present in the temporal evolution of single discs. However, this first requires the development of experimental methods with which single discs can be followed over time (Aegerter-Wilmsen, 2012).

Even though the development of cell shape patterns constitutes a fundamental prediction of the model, it would be an interesting future experimental challenge to test the model's basic assumptions directly, i.e., the regulation of Yki, Arm and ds by mechanical forces. The regulation of Yki by mechanical compression is most relevant for the model's behavior and appears necessary to obtain growth termination in combination with roughly uniform growth. The regulation of Arm by compression seems to be involved in stabilizing the Vg gradient, which could be relatively unstable if it would be regulated by Vg autoregulation alone. In addition, this interaction smoothens the compression gradient, which might have implications for the 3D structure of the wing disc. Last, the regulation of ds by mechanical forces is not essential for the principle behind size regulation, but improves the modeling results and also contributes to smoothening of the compression gradient. While developing the model, focus was placed on its ability to reproduce specific features of growth dynamics, as well as a number of key experiments that are used to argue in favor and against current models. One of the latter results, the decrease of medial growth upon induction of uniform Dpp signaling, could not be reproduced. In the simulations, these discs grow very fast. It is conceivable that such growth rates cannot be sustained in vivo because of a limited availability of nutrients and oxygen. When imposing a maximum total growth rate on disc growth, it is indeed possible to obtain growth rates in the medial part that are lower than those in wildtype discs, whereas lateral growth rates are higher, in agreement with experiments. Thus, with this additional assumption, the model can reproduce the results it was aimed to reproduce (Aegerter-Wilmsen, 2012).

There are currently no experimental data available on the parameters underlying the model and therefore they were fitted manually. As has become clear from the parameter analysis, there are only a few parameter combinations that can reproduce all results. However, it is not known whether this set is reproduced robustly in vivo and there is no natural selection on reproducing experimental manipulations robustly. Nevertheless, it is entirely possible that a larger set of parameter values should reproduce the results. In addition, even though the model can reproduce the selected set of experimentally observed features, there are related observations it cannot reproduce. For example, the final size reached in the model is too small, the experimentally observed nonautonomous growth induction by clones overexpressing brk is nearly absent in the model, and growth induction along the boundary of ds overexpressing clones extends further inside the clone than measured experimentally. It would be interesting to study whether there are factors missing in the model, which would make the parameter space less strict. For example, the parameter space was strongly restricted by the stipulation to reproduce the absence of Vg-BE activity in ap0 mutants upon ectopic wg expression. If it could be assumed that smaller discs have a different geometry in vivo than larger ones, the number of possible parameter combinations would increase. It will be interesting to assess the geometrical properties of discs in young larvae and evaluate whether the model should be adjusted in this respect (Aegerter-Wilmsen, 2012).

Very recently, another model has been formulated for growth regulation that assumes that growth is regulated by increases of Dpp signaling levels over time. However, growth is increased in wing discs in which Brk and Dpp signaling are removed. This either contradicts this model or the current understanding of Dpp signaling needs to be revised. The current model reproduces increased growth in such mutants, including its non-uniformity (Aegerter-Wilmsen, 2012).

The adult wing is covered by bristles, which point towards the distal part of the wing. This orientation is regulated by planar polarity genes. Regulation of planar polarity seems to be related to growth regulation. For example, Ds and Fj are not only important for growth regulation, but are also required for the development of a proximodistal polarity pattern. It is currently not clear whether Ds and Fj are directly involved in regulating planar polarity. If this were the case, then the model would suggest that planar polarity may, at least in part, arise from an interplay between morphogens and mechanical forces. The model presented in this study was developed for the wing imaginal disc of Drosophila. It would be interesting to see whether a similar model could also reproduce size regulation and additional experimental results in other systems. For other imaginal discs, it has been shown that their centers are also compressed at the end of growth. The precise regulatory networks involved in growth and size regulation are different for the different discs, but it would be interesting to see whether certain principles are conserved. In mammals, mechanical forces regulate growth in many tissues. However, the situation is often very different from that in the wing disc in that most mammalian tissues reach their final size while they perform a biological function. Thus, it would be interesting to study whether principles similar to those described here apply for mammalian organs early during development (Aegerter-Wilmsen, 2012).


Dependence of bristle pattern on size and joint formation was studied for male first leg tarsi of fj (four jointed) and d (dachs) mutants in homozygotes and in mosaics resulting from X-ray induced mitotic recombination. Homozygotes have four tarsal segments, lacking a third tarsal joint in most cases. The two proximal segments are shortened, the first by one-third, and altered in bristle pattern, whereas the distal two segments are little affected. Expressivity of fj is high, and of d is low, for the extent and frequency of joint failure. The longer the second segment, the more complete the third joint and the greater the bristle number. Only the jointed side of the segment approximates two segments in its bristle pattern. Mosaic studies show that joint failure occurs autonomously in fj, or in the majority of d clones, and that joint formation by heterozygous clones is autonomous except in the border area contacting a fj or d spot lacking a joint, that is, an area in which a joint failure occurs. Bristle pattern in this jointless heterozygous area switches to that of a single segment. Localized non-autonomy also occurs in the t-rows of heterozygous tissue contacting a fj or d spot. Both mutant genes are interpreted as reducing longitudinal growth of the proximal tarsi, with joint failure as a consequence, and with alterations of bristle pattern resulting directly from size reduction, or indirectly through joint failure (Tokunaga, 1976).

Flies containing the original enhancer trap insertion (Villano, 1995) had no detectable phenotype. To create mutations, the P element insertion was remobilized. All excision lines were subsequently screened by Southern analysis to confirm that phenotypes occur only when excision of the P element is accompanied by deletion of adjacent genomic DNA. All such deletion lines are viable; however, they display a very specific loss of growth in the middle of the tarsus and in the middle portion of the wing blade. The single extant allele of fj (fj1) failed to complement the excision mutations, suggesting that these are mutations in the same gene. Therefore the new mutant lines were named fj2 , fj3 and fj4 (Villano, 1995).

The size and shape of the adult wing are also altered. These changes are largely the result of a shortened separation between the anterior and the posterior crossveins of the wing, an interval that occupies the middle portion of the PD axis in the wing blade. To distinguish between a reduction in cell size versus a reduction in cell number as the origin of this reduced interval, advantage was taken of the observation that each cell of the wing blade produces a single hair, consequently the number and density of hairs in any portion of the wing reflects the number of cells. In this manner, the number of cells along the PD axis on a line parallel to vein 4 between the base of the wing blade (at the junction of veins 4 and 5) and the distal wing margin were counted. It was found that the reduction in inter-crossvein separation seen in the mutant is due to a reduction in total cells in this region, rather than exclusively to a reduced cell size or altered cell shape. As in the leg, dorsal-ventral values appear to be unaltered in the mutant. There is, however, a slight increase in the separation between the wing veins in the AP dimension. This may be an indirect consequence of the global alteration in wing morphology caused by loss of PD growth. Alternatively, fj may have a more subtle but direct effect on growth in the AP axis itself (Villano, 1995).

The eye is the only other tissue visibly affected in the mutants and the phenotypes observed are mild. In strong fj mutants, the external eye shows occasional disordered lens facets and in tangential sections of these eyes a small number of ommatidia (0-33 per eye) appear to be fused with their neighbors so that they are no longer separated by intervening pigment cells. The projections of the photoreceptor neurons to the mutant optic lobes are normal. A weaker allele of fj (fj4) was recovered that gives phenotypes intermediate between those of the strong alleles and wild-type flies. The legs of fj4 adults contain either partial joints at the T2/T3 boundary or double partial joints of like polarity at this position, while a full loss of this joint is rare. Partial joints always occupy the dorsal surface of the leg and showed an incomplete ball and socket invagination, sometimes accompanied by two sensilla campaniformia on the proximal side as in a normal T2/T3 joint. However, the intersegmental membrane on the ventral aspect is absent and the bristle patterns in this area are compressed and distorted slightly on this surface, although bristle pattern elements from each segment are present. This hypomorphic phenotype reinforces the interpretation that fj causes a fusion of T2 and T3 rather than loss of either segment. The crossvein separation in the wing is intermediate between that of fj2 and wild type, while the external eyes appeared to be normal (Villano, 1995).

A deficiency for this region [Df(2R)PC4] uncovers the phenotypes described above with each of the alleles, suggesting these phenotypes result from hypomorphic activity of the gene product. Moreover, the deficiency fails to uncover additional phenotypes so that the restricted loss of PD growth seen in the strong mutants does not appear to result from incomplete loss of gene function (this is supported by a transcript analysis, which shows that fj2 is a null allele) (Villano, 1995).

Several observations suggest that developing ommatidia in the Drosophila eye have distinct dorsal/ventral (DV) positional identities, despite their morphological uniformity. To identify molecular differences along the DV axis of the eye, a systematic screen was carried out for P-element insertions that show nonuniform reporter gene expression along this axis. P-element insertions were identified in which lacZ expression is activated in dorsal, ventral, or equatorial regions of the disc. These patterns of transcriptional enhancer activity are established early in disc development and are maintained in a size invariant manner during disc growth. Several insertions with an equatorial-to-polar gradient of lacZ expression disrupt the four-jointed (fj) gene, which is required for proper leg, wing, and eye development. The fj cDNA sequence includes a presumptive internal signal sequence, indicating that fj encodes a cell surface or secreted protein. Analysis of the fj phenotype and expression pattern in the leg suggests that fj is required for cell-cell signaling during disc development (Brodsky, 1996).

Insect cuticles have been a model system for the study of planar polarity for many years and a number of genes required for this process have been identified. These genes organize the polarized arrangement of hairs on the legs, wings, thorax, and abdomen of adult Drosophila. four-jointed has been shown to be involved in planar polarity decisions in the eye as well as proximal distal leg and wing development. four-jointed is expressed in a gradient through the developing wing and it is required for planar polarity determination in both the wing and the abdomen. Clones of cells either lacking or ectopically expressing four-jointed cause both autonomous and nonautonomous repolarization of hairs in these tissues. It is proposed that the inferred four-jointed expression gradient is important for planar polarity establishment and that local inversions of the gradient by the clones are the probable cause of the observed polarity phenotypes. In addition defects in wing vein development are observed. The subtle phenotypes of mutant flies, and the diverse patterning processes in which it is involved, suggest that four-jointed may act as a modifier of the activity of multiple other signaling factors (Zeidler, 2000).

The wings of fj null individuals were examined for defects in planar polarity. While the normal regular pattern of wing hairs pointing toward the distal end of the wing is essentially unchanged in these individuals, occasional minor defects in wing hair polarity have been identified. A series of misexpression experiments were carried out to test for a link between fj and planar polarity determination. When fj is uniformly expressed using the actin promoter and the Gal4/UAS system occasional examples of wing hair swirls are generated in the proximal regions of the wing. fj is also misexpressed in a stripe along the anterior margin of the anterior/posterior compartment boundary of the developing wing using the patched-Gal4 driver line. When fj is expressed in these cells, nearby wing hairs rotate toward the highest levels of fj expression close to the compartment boundary such that they appear to respond to the combined effect of both the endogenous polarizing activity in the wing and that generated by the ectopic fj (Zeidler, 2000).

Because fj-related phenotypes in the eye are associated with clonal boundaries, and because no suitable clonal markers that do not themselves obscure the polarity of mutant clonal tissue are available, clones lacking fj and marked with yellow were initially generated using the FLP/FRT technique and the amorphic fjd1 allele. The effect of localized removal of fj activity in the wing was found to produce striking disruptions in the normal pattern of wing hair planar polarity and occasionally results in the loss of wing hairs within the presumptive mutant area. The occasional instances in which the yellow marker can be scored indicate that the phenotypes are associated with fjd1 mutant tissue. When plotted together in a composite diagram showing the orientation of disrupted planar polarity associated with clones in many wings, an overall indication of the potential planar polarity phenotypes that can be induced is obtained. This pattern is similar to the phenotype observed in wings from individuals homozygous for hypomorphic fz alleles (Zeidler, 2000).

Loss-of-function mutant fjd1 clones were generated in the wing marked by sha with which the precise boundary of the clone could be marked and any nonautonomous effects observed. While the sha1 allele used has no significant nonautonomous effect, sha1;fjd1 double mutant clones sometimes display striking wing hair planar polarity phenotypes in the wild-type hairs on the proximal side of mutant patches. However, clones smaller than approximately 5x5 cells never give nonautonomous phenotypes. When the positions of clones that produce nonautonomous phenotypes are plotted, only clones present within certain areas are found to generate nonautonomous polarity phenotypes. All clones larger than approximately 5x5 cells produce nonautonomous phenotypes in a region distal to the posterior cross vein. Larger clones also produce strong nonautonomous phenotypes in a region between veins 3 and 4, and weaker phenotypes are observed in a large region in the center of the wing posterior to vein 4. The positions were plotted in which polarity phenotypes induced by unmarked clones were seen, and this was seen to extend throughout almost the entire wing blade, in marked contrast to the limited area in which nonautonomous phenotypes are seen (Zeidler, 2000).

While the severity and extent of the phenotypes generated depend on clone position and size, the inversion of wing hair polarity is consistently seen on the proximal side of clones, resulting in the hairs pointing from a region lacking fj expression (in the clone) to regions of higher level fj expression (in the proximal wild-type tissue). Thus both misexpression and loss-of-function experiments are consistent with wing hairs pointing towards high levels of Fj. It should also be noted that the swirls of hairs associated with the wild-type tissue proximal to sha1;fjd1 double clones have a tendency to point toward the posterior of the wing. This result appears to be consistent with the composite results obtained from unmarked fjd1 single-mutant clones and may be a result of the higher levels of fj expressed at the posterior margin of the wing (Zeidler, 2000).

As in the wing, planar polarity in the Drosophila abdomen is also exhibited by the polarity of hairs and bristles present on the cuticle of much of each abdominal segment. Unlike in the wing, in which only negligible polarity defects are observed, fj homozygotes show hair polarity defects in the abdomen, albeit in a very limited region of each segment. In the a2 region, for about 50% of fjd1 abdominal segments, the hairs show extensive 'swirls' in a restricted region close to the midline, with weaker phenotypes observed in almost all segments. In addition, two different classes of loss-of-function clones were generated. First sha1;fjd1 double mutant clones were generated to identify nonautonomous phenotypes. Interestingly, fj does not appear to generate planar polarity phenotypes in the a4, a5, or posterior regions of a3. However, clones positioned in a2 and the anterior of a3 do show clear nonautonomous hair inversions adjacent to the posterior margin of mutant tissue. While sha-marked clones mark the extent of mutant clones, this method precludes the analysis of planar polarity within mutant regions. Double-mutant clones lacking the body pigment marker yellow and fjd1 were therefore induced such that yellow mutant bristles in the abdominal cuticle marked the position (but not the precise outline) of the clones. In contrast to the situation in the wing, autonomous phenotypes are found in essentially the same region as the nonautonomous effects described above. Clones in a3-a5 show no phenotype, while clones in anterior a3 result in major disruptions of hair polarity. Given the greater range of inversions apparent in yellow- versus sha-marked fjd1 mutant clones, it is clear that the majority of inverted hairs are present within mutant tissue and that inversions are not restricted to adjacent wild-type cells (Zeidler, 2000).

Since the loss-of-function clonal analysis indicates that fj may be required only in a relatively small portion of the abdomen, the regions in which fj misexpression is sufficient to generate repolarization using an actin-Gal4 driver line activated by removal of a yellow1 'stuffer' element were generated by FLP recombinase. In this way actin-Gal4/UAS-fj-misexpressing, yellow-marked clones could be induced in the abdomen (Zeidler, 2000). Clones present in all regions in which the yellow marker could be scored (a3-a5) generate polarity inversions at their anterior margins. In addition the darker pigmentation present in the a5 region makes it possible to determine the exact outline of the fj-misexpressing, yellow-marked clone, demonstrating that inversions can extend nonautonomously over at least seven rows of wild-type hairs (Zeidler, 2000).

The results presented regarding hair polarization in the wing and abdomen are intriguing as it appears that wing hairs point towards high fj activity and that abdominal hairs are oriented away from clones of high fj expression. This illustrates that while fj can polarize hairs in both abdomen and wing, the direction of polarization is not invariant and can change from tissue to tissue (Zeidler, 2000).

In addition to the role of fj in planar polarity determination demonstrated here, mutations in the locus are associated with both the fusion of tarsal joints and the proximal-distal (PD) shortening of both leg and wing structures. During these investigations it became clear that many fj misexpression experiments generate PD phenotypes. The phenotypes associated with loss of fj were compared to those produced by uniform fj misexpression using the actin-Gal4/UAS-fj system. In comparison to legs from wild-type flies, legs from fjd1 homozygotes are clearly shortened and display the characteristic fusion of the second and third tarsi. Defects in the legs of actin-Gal4/UAS-fj individuals are very similar and include PD axis shortening as well as tarsal fusion. While actin-Gal4-driven defects are striking, the ectopic fj expression driven by the patched-Gal4 line can produce much more severe phenotypes and can result in extreme PD axis shortening and loss of distal structures while the coxa, a proximal structure, appears largely unaffected (Zeidler, 2000).

Wild-type wings are longer than those lacking fj activity and those in which fj is uniformly expressed by the actin-Gal4/UAS-fj system. In order to better classify the phenotypes obtained, the lengths of different sections of the wing were measured and compared. This shows that while proximal regions of the wing are unaffected the region between the anterior and the posterior cross vein is significantly reduced in both the null mutant (fjd1) and the enhancer detector P-element insertion (fjP1) homozygotes as well as in the actin-Gal4/UAS-fj-misexpressing individuals. While the effect on more proximal structures is more variable the overall wing lengths are significantly reduced in all cases. Thus actin-Gal4/UAS-fj produces PD defects both qualitatively and quantitatively similar to those produced from the total removal of fj in a homozygous amorphic situation. In addition to the previously characterized role of fj in leg outgrowth fj mutant individuals were also observed to have shorter and 'dumpier' abdominal regions than wild type. In order to quantitate this observation the width of the region of abdominal segments between the a1/a2 and the a5/a6 boundaries was quantitated. While wild-type abdomens are an average of 261 mm at the dorsal midline, fjP1 homozygous abdomens are 221 mm in width. Other fj mutants show similar effects. Thus it is clear that the contraction of PD growth observed in the leg and wing also occurs in the abdomen (Zeidler, 2000).

A number of additional observations also indicate a link between fj and the patterning of wing veins. Beginning at about 18 h APF down-regulation of fj enhancer trap expression is seen in longitudinal stripes in the wing, which ultimately resolve to a pattern that is thought to represent the future veins. In addition to this, wings homozygous mutant for fj often contain ectopic wing vein material as do fjd1 mutant clones in which vein material forms within or adjacent to the clone. Furthermore misexpression by actin-Gal4/UAS-fj also produces wing vein phenotypes including loss of the posterior cross vein and displacement of vein 4 at the junction with the wing margin as well as the deletion of the anterior cross vein in the region of fj misexpression in patched-Gal4/UAS-fj wings. In some sha1;fjd1 double-mutant clones, veins are often diverted or duplicated around mutant regions so that the vein material is situated in wild-type tissue immediately abutting the clonal boundary and in some cases veins 'fade' in mutant tissue. These results imply that fj acts as a regulator of wing vein formation and suggest that in normal development fj might function to ensure the precise positioning of the veins (Zeidler, 2000).

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).

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).

Previous studies have demonstrated that four-jointed (fj), which encodes a type II transmembrane/secreted protein, is expressed in a graded fashion from the equator toward the poles of the eye imaginal disc. Fj has been implicated in the regulation of ommatidial polarity based on the observation that reversals of ommatidial polarity occur along the polar border of fj mutant clones. However, fj mutant flies and the interior of fj clones only rarely display polarity defects. This has suggested that much of Fj action during ommatidial polarity formation may be redundant with other signals. The role of Fj was investigated further by asking whether Fj might regulate Ds or Ft function. To determine whether Fj acts predominantly in one member of the R3/R4 precursor pair, strong loss-of-function fj mutant (fjN7) clones were generated, and R3/R4 mosaic ommatidia were examined. The loss of Fj function from the polar precursor cell, which expresses lower levels of Fj, had little effect on ommatidial polarity (6% reversals in 70 cases). In contrast, loss of Fj function from only the equatorial precursor cell led to polarity reversals in 71% of the 52 cases. These results indicate that Fj is mainly required in the equatorial R3/R4 precursor cell during the determination of ommatidial polarity. However, when only the equatorial cell lacks functional Fj, the Fj produced by the polar cell frequently is able to reverse the pattern of R3/R4 specification. These data suggest that the Fj gradient may play a role in directing R3/R4 specification by providing more Fj activity to the equatorial precursor cell (Yang, 2002).

The frequent polarity reversals caused by loss of Fj from the equatorial cell contrasts markedly with the paucity of polarity defects in fj mutant animals. A possible explanation for this difference is that Fj might act by modulating Ds function. Thus, loss of Fj from an entire eye would leave a Ds gradient in place to direct ommatidial polarity. In contrast, loss of Fj from only the equatorial precursor may reverse the gradient of Ds function within an R3/R4 pair. If this model were correct, differences in Fj function between the R3/R4 precursor cells should be ineffective at biasing cell fate specification in the absence of Ds. However, differences between the R3/R4 precursor cells in their levels of Ds function should be able to direct ommatidial polarity even in the absence of Fj (Yang, 2002).

The first prediction was tested by examining fj clones generated in dsUA071 flies. Since the lack of a recognizable equator in homozygous ds mutant eyes made it impossible to designate normal versus reversed polarity ommatidia in fjN7 clones, the fj R3/R4 mosaic ommatidia were analyzed by measuring the frequency with which the fj-/- precursor cell became R4. When fj mosaic ommatidia were assayed in ds mutant flies, the fj-/- precursor cell was equally likely to become R3 or R4 (54% as R4, 46% as R3). In contrast, the fj-/- cell became R4 in 84% of cases when fj mosaic clones were generated in a ds+ background. These data show that differences in Fj function can only specify the pattern of R3/R4 specification when Ds is present. The ability of differences in Ds levels to influence R3/R4 specification in the absence of Fj function was examined by generating dsUA071 clones in either wild-type or homozygous fjN7 flies. When ds mutant clones were induced in wild-type background, the ds-/- cell has 68% chance of becoming an R3. Interestingly, when ds clones were induced in fj animals, this frequency was increased to 85%, showing that the lack of Fj not only fails to block the action of Ds but instead enhances the ability of Ds differences to dictate the pattern of R3/R4 specification. These results support a model in which Fj acts upstream of Ds, perhaps as a regulator of Ds function (Yang, 2002).

Since fj and ds are expressed in opposing gradients in the eye, one possible mechanism by which Fj could regulate Ds function would be to inhibit ds transcription. This possibility was investigated by examining ds-lacZ expression in clones of cells that either lack Fj function or overexpress Fj. The lack of detectable effect on ds-lacZ expression in these experiments indicates that Fj does not act by antagonizing ds expression (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 armH8.6 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).

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).

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).

Four-jointed and abdominal compartmentalization

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 interest to understand 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 had 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).

Fj is a type II transmembrane glycoprotein that may be cleaved and secreted. Both in the fly eye and in the wing, it is expressed in a graded manner. Studying clones ectopically expressing fj in the wing, it has been concluded that the orientation of hairs is reversed, distal to some clones, and this finding suggests that neighboring cells point toward the ectopic source of Fj, that is, up the presumed gradient of Fj. However, the abdomen, where cells make hairs that point posteriorly, has also been examined. Here, clones expressing Fj reverse the polarity of abdominal hairs anterior to the clone, as if the hairs were pointing away from the source of ectopic Fj and down the presumed gradient. The results on the wing and abdomen, therefore, appear to differ in sign (Casal, 2002 and references therein).

To find where Fj is expressed relative to the A and P compartments of the abdomen, a fj.lacZ transgene was used. In the dorsal epidermis, which forms the tergite, expression is concentrated both in the bristled portion of the A compartment (the a3–a5 territories) and in the most anterior portion of the A compartment (a1–a2). Strong expression is observed in the sternite, which also forms bristles. However, the remainder of the ventral epidermis forms pleura, a lawn of cells that secretes only hairs, and here the pattern of expression is simple: there is a band of staining near the front of the A compartment. One attractive interpretation is that the fj.lacZ transgene may pick up extraneous enhancers active in bristly (neurogenic) cuticle; therefore, the 'true' Fj pattern is seen only in the nonneurogenic pleura. This is attractive because it is consistent with the following independent data suggesting that there is a gradient of Fj activity, with its peak at the front of A (Casal, 2002).

Flies that lack fj activity in all cells show some effects on pattern. In the abdomen, there is some dishevelment of hairs and bristles, but only in the anterior portion of the A compartment (in a2 and a3). It seems, therefore, that planar polarity is specified almost normally in the absence of Fj (Casal, 2002).

The clones were labelled genetically so that each cell of the clone could be distinguished from its neighbors. Within the A compartment, fj- clones are abnormal, but only when they are located in approximately the front half of the compartment. Each cell typically produces little groups of posteriorly pointing hairs arranged in neat mediolaterally oriented rows, but within affected fj- clones, the rows of hairs are jumbled. Also, the hair orientation is disturbed, with most of those at the back of the clone, and the wild-type hairs behind it, being reversed. It seems that, the further anterior the clone, the more disturbance within and the more reversal of hair polarity behind. In the posterior part of the A compartment, removing fj from clones has no effect on the orientation and arrangement of hairs; all such clones are exactly like controls in which only the marker gene, pwn, is missing (Casal, 2002).

The effects of fj- clones on polarity in the P compartment can be assayed only in the p3 territory -- because only p3 cells make hairs, while p2 and p1 cells are bald. Some clones in the p3 territory form dishevelled and incorrectly polarized hairs; however, most appear normal. No clear cut cases of non-autonomous effects were found outside these P clones. Note that the p3 region is remote from the presumed peak of Fj near the front of the A compartment (Casal, 2002).

Thus, endogenous Fj activity production appears to be required in the front half of the A compartment. In this region, clones of fj mutant cells cause surrounding cells to make hairs that point inward, and this suggests that the hairs point down the gradient of Fj protein (Casal, 2002).

Various Gal4/UAS and G80 techniques were used to make marked clones of cells that produce ectopic Fj protein. Different levels of Fj expression were achieved with two Gal4 drivers of different strengths. In the A compartment, clones of cells in which is expressed under the control of the weaker driver (abx/ubx.Gal4) cause a polarity phenotype when they are located in the back of A, but not when they are elsewhere. Within the clone, there are whorls, and these can extend outside the clone in the anterior, but not the posterior, direction. Note that this is the opposite of the phenotype in fj- clones in two ways: (1) clones cause polarity changes if they are at the back of the A compartment, while fj- clones cause changes only at the front; (2) fj- clones cause a reversal of polarity behind the clone, while clones alter polarity in front. Clones within the P compartment appear normal (Casal, 2002).

With the stronger driver (tub.Gal4), the phenotype is more definite; there are few whorls, and, instead, the hairs are reversed within the anterior part of the clone, and this reversal extends anterior to the clone itself. These non-autonomous effects can spread as much as 6 or 7 cell diameters. This effect is found over most of the A compartment and includes clones in the anterior region of the bristled cuticle (a3) that cause extensive reversal anterior to them (in a2). However, some clones within the extreme anterior portion of the a2 region cause little or no changes in polarity anterior to the clone. This difference could be because cells at the extreme anterior of A normally make a large amount of Fj protein, so overexpressing the gene there might have little impact on the landscape of concentration. Thus, the behavior of A clones that lack or overexpress fj suggests that changes of polarity are induced wherever there is a difference in levels of Fj between the clone and its immediate neighborhood. All the results fit nicely with this idea and argue that there is normally a gradient of Fj that is high at the front of A and low at the back (Casal, 2002).

In the P compartments, these clones are also associated with polarity reversals. But here, consistently, hairs within the back half of the clone as well as hairs behind are reversed and now point anteriorly. This contrasts with effects of clones located at the other side of the A/P boundary in the posterior of the A compartment (in the a6, a5, a4, and a3 territories). In this case, hairs in the anterior half of the clone, as well as in front of the clone, point anteriorly. Thus, in both cases, polarity reversals are observed in territories farthest from the apparent source of Fj activity; however, in the A compartment, hairs point away from the ectopic source, whereas, in the P compartment, hairs point toward the source. These results support a model in which the A and P compartments have opposing gradients of Fj activity, and cells within each compartment are programmed to respond to the vector of Fj activity by secreting hairs that point down the gradient in A but up the gradient in P (Casal, 2002).

Clones situated near the boundaries between the A and P compartments raise new problems. Consider first clones at the back of the A compartment -- the interface between posterior A and anterior P cells. These clones reverse the hairs in front of the clone, which would be normal for clones in the back half of A. The back of the clone is itself made of A cells; however, they abut P cells behind them, and these P cells are also reversed. Apparently, polarity effects (and maybe the ectopic Fj protein) can cross over the parasegment boundary from A to P (Casal, 2002).

Now consider clones at the front of P; these clones would be expected to reverse the P cells behind them, and they do. However, they might also be expected to reverse the cells in the A compartment in front of them, but they do not. Perhaps polarity effects crossing over from P to A are blocked. There is some evidence that normally, Hh induces cells in posterior A (say, the a6 region) to sequester and/or destroy ambient Fj. This would create a local sink for Fj at the point farthest from its source and help build the gradient of Fj. Overexpression of fj at the back of A might make enough protein to overcome this sink, thus creating an ectopic peak of Fj extending both anteriorly into A and posteriorly across the A/P boundary into the P compartment, with consequent polarity reversal in both compartments. By contrast, overexpression of Fj in P (or by just one row of cells at the back of A) might not generate sufficient Fj across the boundary to overwhelm the sequestering activity of A cells, so only cells in P would see an ectopic peak and be repolarized (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 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).

But this new model raises yet another challenge: consider the pleura, which is formed by a sheet of cells spanning several segments, all of which secrete hairs that point posteriorly. If, for example, the localization of Fz in each cell were controlled by the graded activity of Ft, then these Ft gradients would need to be precisely coextensive with the compartments. Otherwise, some cells would read gradients with the wrong sign and make hairs that point in the wrong direction. This could be most critical at the boundaries between the A and P compartments, where the gradient landscape of Ft should be forming peaks or troughs and hence might be relatively flat. This challenge could be resolved if, in a later and/or independent process, cell polarity were locally coordinated: there is some evidence for this. For example, clones of cells that lack or overexpress Fz can cause local reversals in hair polarities that propagate a few cell diameters beyond the clone borders (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).

Mutations that cause a reduction in cell division are common, but those, such as ft, that cause increased growth are rare. The ft gene may be a link between planar polarity and growth — it has been suggested that a morphogen gradient may control both. If the slope or vector of a morphogen is used to specify planar polarity, the local steepness of that same gradient might provide a measure of dimension. This measure would then help determine the probability of cell division and apoptosis, regulate the rate of net growth, and limit the final size (Casal, 2002).

Boundaries of Dachsous Cadherin activity modulate the Hippo signaling pathway to induce cell proliferation

The conserved Hippo tumor suppressor pathway is a key signaling pathway that controls organ size in Drosophila. To date a signal transduction cascade from the Cadherin Fat at the plasma membrane into the nucleus has been discovered. However, how the Hippo pathway is regulated by extracellular signals is poorly understood. Fat not only regulates growth but also planar cell polarity, for which it interacts with the Dachsous (Ds) Cadherin, and Four-jointed (Fj), a transmembrane kinase that modulates the interaction between Ds and Fat. Ds and Fj are expressed in gradients and manipulation of their expression causes abnormal growth. However, how Ds and Fj regulate growth and whether they act through the Hippo pathway is not known. This study reports that Ds and Fj regulate Hippo signaling to control growth. Interestingly, it was found that Ds/Fj regulate the Hippo pathway through a remarkable logic. Induction of Hippo target genes is not proportional to the amount of Ds or Fj presented to a cell, as would be expected if Ds and Fj acted as traditional ligands. Rather, Hippo target genes are up-regulated when neighboring cells express different amounts of Ds or Fj. Consistent with a model that differences in Ds/Fj levels between cells regulate the Hippo pathway, it was found that artificial Ds/Fj boundaries induce extra cell proliferation, whereas flattening the endogenous Ds and Fj gradients results in growth defects. The Ds/Fj signaling system thus defines a cell-to-cell signaling mechanism that regulates the Hippo pathway, thereby contributing to the control of organ size (Willecke, 2008).

These data show that Ds and Fj regulate the Hippo pathway in an unusual manner. Most interestingly, it was found that discontinuities or boundaries of Ds and Fj activity, rather than their absolute amounts, modulate the Hippo pathway. The effects of Ds and Fj on wg expression in the hinge region are also consistent with the proposed boundary model. Importantly, artificial Ds/Fj boundaries cause an up-regulation (de-repression) of Hippo pathway target genes and drive extra cell proliferation, whereas flattening of the endogenous Ds and Fj gradients reduced normal growth. Together, these data are consistent with a model in which Ds/Fj discontinuities suppress the activity of the Hippo pathway, thereby driving imaginal disc growth and thus contributing to the regulation of organ size (Willecke, 2008).

How much growth is controlled by Ds/Fj signaling? Flies with uniform Ds/Fj expression have significantly reduced wings, legs, and other body parts, but growth is not abolished. The Ds boundary effect thus accounts for some but not all growth control. Given that fat mutants have severely overgrown imaginal discs, how do these growth defects caused by flat Ds/Fj expression fit with a model that Ds and Fj act through Fat to regulate growth? The dachs mutant phenotype gives insights into that question. Dachs acts downstream of Fat and is required for the growth control function of Fat. Unlike Fat, however, Dachs is a positive regulator of growth. Fat thus suppresses growth by inhibiting Dachs, and the dachs mutant phenotype thus reflects the situation where Fat is fully (hyper) active. because Fat functions through the inactivation of Dachs, the growth defects caused by Fat hyperactivation cannot be stronger than the dachs mutant phenotype. The boundary model proposes that flattening the Ds and Fj gradients results in hyperactivation of Fat, thereby causing reduced growth. Remarkably, dachs mutants have small wings and short legs, and the strength of these growth defects are similar to those caused by uniform Ds and Fj expression. The phenotypes caused by uniform Ds and Fj expression are thus consistent with the model that discontinuities of Ds and Fj inactivate Fat signaling to promote growth (Willecke, 2008).

The observation that flies with uniform Ds/Fj expression as well as dachs mutants retain some growth indicates that other signaling mechanisms act in addition to the Ds boundary effect to control imaginal disc size. The Ds boundary effect is thus one of possibly several separate mechanisms that contribute to control the final size of imaginal discs. These other, currently unknown signals may act in parallel to the Hippo pathway to regulate tissue growth. In addition, other signals may regulate the Hippo pathway independently of the Ds boundary effect. For example, Mer acts in parallel to Fat, thus identifying another input into the Hippo pathway. It will be interesting to elucidate these additional signaling systems and to understand how they cooperate with Hippo signaling to control imaginal disc growth (Willecke, 2008).

Ds, Fj, and Fat regulate growth and planar cell polarity (PCP). Interestingly, discontinuities in Ds/Fj activity rather than their absolute amounts also regulate PCP. Ommatidial polarity reversals are associated with ds and fj mutant clone borders and ommatidia inside and outside of clones are affected. Boundary effects of Ds and Fj are also observed on hair polarity in the wing and abdomen. Thus, Ds/Fj discontinuities modulate Hippo signaling and PCP. However, the effects on the Hippo pathway are different from those on PCP. In contrast to the effects on Hippo target genes, which are induced all around clone borders, PCP effects are observed only on one side of clones. This difference can be explained because the Hippo readout is scalar (levels of target gene expression), whereas the PCP readout is vectorial (direction of polarity). Thus, the direction of the Ds/Fj gradients determines the direction of cell polarity, whereas the disparity in Ds/Fj activity (steepness of the gradients) modulates Hippo signaling (Willecke, 2008).

The effect of Ds/Fj boundaries appears to spread over several cells. Although β-Gal perdurance may contribute to this effect when assaying reporter gene expression, it was found that Ex degradation as well as up-regulation of DIAP1 protein, which has a short (30-min) half-life, is also observed over several cell diameters, indicating that the boundary signal is propagated over several cells. A similar propagation is also observed for the effects of Ds/Fj boundaries on PCP, and it has been suggested that Ds/Fj boundaries cause an asymmetric localization of Ds and Fat which may then propagate between cells (Willecke, 2008).

The boundary model proposes that cells respond to disparities in the levels of Ds/Fj between cells. How do cells sense Ds/Fj disparities to modulate downstream effectors? Ds forms heterodimers with Fat on neighboring cells and Fat cell autonomously regulates the activity of the Hippo pathway. This suggested that Fat and Ds may act as receptor and ligand, respectively. Surprisingly, however, Ds and Fat do not behave like a classical ligand-receptor pair. First, Ds does not regulate Fat in a dose-dependent manner, but rather acts through a boundary effect. Second, Ds is required in signal-sending cells as well as in responding cells, indicating that Ds has ligand- and receptor-like functions. This is true for the regulation of Hippo signaling as well as for PCP signaling in the abdomen. The finding that the intracellular domain of Ds is not required for the generation but for the sensing of the boundary signal further exposes this dual function of Ds. Two alternative models could explain how cells sense Ds discontinuities. In a first model, cells may compare the amount of Ds presented by neighboring cells on opposite sides. Cells may then sense a differential in the number of bound Fat molecules from one side of the cell to the other. In an alternative model, cells may compute the difference between the amount of Ds presented by neighboring cells (sensed by the amount of bound Fat molecules) with the amount of Ds expressed by a cell itself. This model may explain why Ds and its intracellular domain are required cell-autonomously to respond to the boundary signal. In both models, a differential in Ds activity between cells may regulate the activity of Fat, which then transduces the signal to downstream components. Fat is cell-autonomously required to regulate the Hippo pathway, and the intracellular domain of Fat is sufficient to promote the growth control and at least some PCP functions of Fat. The intracellular domain of Fat may thus transduce the boundary signal to downstream components regulating PCP and Hippo. Because Ex, Dachs, Hpo, Wts, and Yki do not or only slightly affect PCP, Fat may engage different downstream effectors to regulate PCP and the Hippo pathway. It will be fascinating to decipher the molecular mechanisms of how boundaries of Ds/Fj activity regulate the activity of Fat and how they are translated into a vector to control PCP and a scalar to modulate the Hippo pathway (Willecke, 2008).

Prickle/spiny-legs isoforms control the polarity of the apical microtubule network in planar cell polarity

Microtubules (MTs) are substrates upon which plus- and minus-end directed motors control the directional movement of cargos that are essential for generating cell polarity. Although centrosomal MTs are organized with plus-ends away from the MT organizing center, the regulation of non-centrosomal MT polarity is poorly understood. Increasing evidence supports the model that directional information for planar polarization is derived from the alignment of a parallel apical network of MTs and the directional MT-dependent trafficking of downstream signaling components. The Fat/Dachsous/Four-jointed (Ft/Ds/Fj) signaling system contributes to orienting those MTs. In addition to previously defined functions in promoting asymmetric subcellular localization of 'core' planar cell polarity (PCP) proteins, this study found that alternative Prickle (Pk-Sple) protein isoforms control the polarity of this MT network. This function allows the isoforms of Pk-Sple to differentially determine the direction in which asymmetry is established and therefore, ultimately, the direction of tissue polarity. Oppositely oriented signals that are encoded by oppositely oriented Fj and Ds gradients produce the same polarity outcome in different tissues or compartments, and the tissue-specific activity of alternative Pk-Sple protein isoforms has been observed to rectify the interpretation of opposite upstream directional signals. The control of MT polarity, and thus the directionality of apical vesicle traffic, by Pk-Sple provides a mechanism for this rectification (Olofsson, 2014).

A model is proposed for coupling Ft/Ds/Fj to the core module. Gradients of Fj and Ds, by promoting asymmetric distribution of Ft/Ds heterodimers, align a parallel network of apical MTs. Vesicles containing Dsh are transcytosed towards MT plus-ends. In the presence of Pk, MT plus-ends are biased towards the high end of the Fj gradient and the low end of the Ds gradient, whereas in the presence of Sple, the MT plus-ends are biased towards areas with low levels of Fj and high levels of Ds expression. Predominance of Pk or Sple, therefore, determines how tissues differentially interpret, or rectify, the Ft/Ds/Fj signal to the core module. It is hypothesized that this signal serves to both orient the breaking of initial symmetry and to provide continual directional bias throughout polarization. Additional validation of this model would require the measurement of Eb1::GFP comet directions while controlling Pk-Sple isoform expression in wings bearing ectopic Ds and Fj gradients, an experiment that is beyond the technical capabilities with currently available reagents. However, further evidence in support of this model is found in the observation that, in Pk-predominant wings, MT polarity and hair polarity point from regions with high toward low Ds expression both in wild-type wings and in wings with ectopic reversed Ds gradients (Olofsson, 2014).

It is noted that the distal plus-end bias of MTs is seen in much of the wild-type wing, but this bias decreases to equal proximal-distal plus-end distribution near to the most distal region of the wing. Thus, the mechanism described in this study might not affect the entirety of the wing; in contrast, plus-end bias was observed across the entire A-abd compartment (Olofsson, 2014).

A model incorporating early Sple-dependent signaling and late Pk-dependent signaling has been proposed to explain PCP in the wing. The current observations and model are compatible with the data presented in support of that model; Sple expression, although always lower than Pk expression in wild-type wing, declined during pupal wing development, suggesting that, in pk mutants, polarity patterns might be set early in development, when Sple is still expressed and when Ds is present in a stripe through the central part of the wing, giving rise to anteroposterior oriented patterns (Olofsson, 2014).

Pk (and presumably Sple, in Sple dependent compartments) is required for amplification of asymmetry by the core PCP mechanism (Tree, 2002; Amonlirdviman, 2005). These results indicate an additional, core module independent, function for these proteins in regulating the polarity of MTs. Furthermore, although the core function of Pk-Sple is not well defined, part of that function might include promoting the formation and movement along aligned apical microtubules of Fz-, Dsh- and Fmi-containing vesicles (Shimada, 2006). The relative abundance of transcytosing vesicles in Pk versus Sple tissues suggests that if Sple promotes MT-dependent trafficking, it does so less efficiently than Pk (Olofsson, 2014).

These activities are remarkably similar to those that have been recently identified for Pk and Sple in fly axons, where Pk promotes or stabilizes MT minus-end orientation towards the cell body, and Sple promotes the orientation of minus-ends toward the synapse, which has effects on vesicle transport and neuronal activity. A common mechanism of differentially adapting the plus- and minus-ends of MT segments is proposed in both instances. In axons, similar to what was observed in this study, Pk also facilitates more robust cargo movement, whereas movement is less efficient when Sple is the dominantly expressed isoform. Furthermore, MT polarity defects might underlie the apical-basal polarity defects and early lethality of mouse prickle1 mutant embryos. As Ft and Ds are not known to regulate MTs in axons, these observations suggest that Pk and Sple are able to modify MT polarity independently of Ft/Ds. However, in wings, a consequence is only evident if MTs are first aligned by Ft/Ds activity (Olofsson, 2014).

How Pk and Sple modulate the organization of MTs remains unknown, but possibilities include modifying the ability of Ft or Ds to capture or nucleate MTs, or altering plus-end dynamics to inhibit capture. These data also suggest the possibility of a more intimate link between the core PCP proteins and Ft/Ds than has been appreciated previously. Other concurrent signals, such as that proposed for Wnt4 and Wg at the wing margin, cannot be ruled out. However, the observations that (1) MTs correlate with the direction of core PCP polarization over space and time, (2) vesicle transcytosis is disrupted in ft clones in which MTs are randomized, (3) chemical disruption or stabilization of MTs disturbs polarity and (4) Pk and Sple isoform predominance rectifies signal interpretation by the core module in a fashion that follows both the wild-type and ectopic Ds gradients provide additional evidence for the model that a signal from the Ft/Ds/Fj system orients the core PCP system in substantial regions of the wing and abdomen (Olofsson, 2014).


The cloning of the mouse four-jointed1 gene is reported and its pattern of expression in the brain during embryogenesis and in the adult. In the neural plate, fjx1 is expressed in the presumptive forebrain and midbrain, and in rhombomere 4, however a small rostral/medial area of the forebrain primordium is devoid of expression. Expression of fjx1 in the neural tube can be divided into three phases. (1) In the embryonic brain fjx1 is expressed in two patches of neuroepithelium: the midbrain tectum and the telencephalic vesicles. (2) In fetal and early postnatal brain, fjx1 is expressed mainly by the primordia of layered telencephalic structures: cortex (ventricular layer and cortical plate), and olfactory bulb (subependymal layer and in the mitral cell layer). In addition, expression is observed in the superior colliculus. (3) In the adult, fjx1 is expressed by neurones evenly distributed in the telencephalon (isocortex, striatum, hippocampus, olfactory bulb, piriform cortex), in the Purkinje cell layer of the cerebellum, and numerous medullary nuclei. In the embryo, strong expression can further be seen in the apical ectodermal ridge of fore- and hind-limbs, and in the ectoderm of the branchial arches (Ashery-Padan, 1999).

The murine sequence has an open reading frame encoding a polypeptide of 437 amino acids. The calculated relative molecular mass of the conceptual translation product is 48,912 Da, and its isoelectric point would be 10.55. Comparison of the predicted amino acids sequence with the currently available database entries reveal significant homology to the Drosophila Fj protein (50% similarity, 42% identity). In addition to sequence similarity, Fj and Fjx1 share structural motives; a putative transmembrane domain, and a signal sequence cleavage site are located in the N-terminal end of both proteins. In the C-terminal region of both proteins, consensus sites for asparagine linked glycosylation are present. In addition, a consensus sequence for alpha amidation, a common modification in proteolytically processed neuropeptides, is predicted in Fj and Fjx1. Finally, the predicted topology of both proteins implies that the C-terminal region is processed in the endoplasmic reticulum and secreted. This is in agreement with in vitro experimental evidence showing that the Drosophila Fj is glycosylated and secreted (Ashery-Padan, 1999).


Search PubMed for articles about Drosophila four-jointed

Aegerter-Wilmsen, T., Heimlicher, M. B., Smith, A. C., de Reuille, P. B., Smith, R. S., Aegerter, C. M. and Basler, K. (2012). Integrating force-sensing and signaling pathways in a model for the regulation of wing imaginal disc size. Development 139: 3221-3231. Pubmed: 22833127

Ambegaonkar, A. A., Pan, G., Mani, M., Feng, Y. and Irvine, K. D. (2012). Propagation of Dachsous-Fat planar cell polarity. Curr Biol 22: 1302-1308. PubMed ID: 22727698

Ashery-Padan, R., et al. (1999). Fjx1, the murine homologue of the Drosophila four-jointed gene, codes for a putative secreted protein expressed in restricted domains of the developing and adult brain. Mech. Dev. 80(2): 213-7. PubMed Citation: 10072791

Boedigheimer, M. and Laughon, A. (1993). expanded: a gene involved in the control of cell proliferation in imaginal discs. Development 118: 1291-1301. PubMed Citation: 8269855

Brittle, A., Thomas, C. and Strutt, D. (2012). Planar polarity specification through asymmetric subcellular localization of Fat and Dachsous. Curr. Biol. 22(10): 907-14. PubMed Citation: 22503504

Brodsky, M. H. and Steller, H. (1996). Positional information along the dorsal-ventral axis of the Drosophila eye: graded expression of the four-jointed gene. Dev. Biol. 173(2): 428-46. PubMed Citation: 8606003

Bryant, P. J., Huettner, B., Held, L. I., Ryerse, J. and Szidonya, J. (1988). Mutations at the fat locus interfere with cell proliferation control and epithelial morphogenesis in Drosophila. Dev. Biol. 129: 541-554. 88329493

Buckles, G. R., et al. (2001). four-jointed interacts with dachs, abelson and enabled and feeds back onto the Notch pathway to affect growth and segmentation in the Drosophila leg. Development 128: 3533-3542. 11566858

Casal, J., Struhl. G. and Lawrence, P. A. (2002). Developmental compartments and planar polarity in Drosophila. Curr. Biol. 12: 1189-1198. 12176328

Cho, E. and Irvine, K. D. (2004). Action of fat, four-jointed, dachsous and dachs in distal-to-proximal wing signaling. Development 131: 4489-4500. 15342474

Cho, E., Feng, Y., Rauskolb, C., Maitra, S., Fehon, R. and Irvine, K. D. (2006). Delineation of a Fat tumor suppressor pathway. Nat. Genet. 38(10): 1142-50. 16980976

Garoia, F., et al. (2000). Cell behaviour of Drosophila fat cadherin mutations in wing development. Mech. Dev. 94: 95-109. 10842062

Ishikawa, H. O., Takeuchi, H., Haltiwanger, R. S. and Irvine, K. D. (2008). Four-jointed is a Golgi kinase that phosphorylates a subset of cadherin domains. Science 321(5887): 401-4. PubMed citation: 18635802

Mahoney, P. A., Weber, U., Onofrechuk, P., Biessmann, H., Bryant, P. J. and Goodman, C. S. (1991). The fat tumor suppressor gene in Drosophila encodes a novel member of the cadherin gene superfamily. Cell 67: 853-868. PubMed Citation: 1959133

Mangione, F. and Martin-Blanco, E. (2018). The Dachsous/Fat/Four-Jointed pathway directs the uniform axial orientation of epithelial cells in the Drosophila abdomen. Cell Rep 25(10): 2836-2850. PubMed ID: 30517870

Mao, Y., et al. (2006). Dachs: an unconventional myosin that functions downstream of Fat to regulate growth, affinity and gene expression in Drosophila. Development 133(13): 2539-51. PubMed Citation: 16735478

Neumann, C. J. and Cohen, S. M. (1996). Distinct mitogenic and cell fate specification functions of Wingless in different regions of the wing. Development 122: 1781-1789. 8674417

Olofsson, J., Sharp, K. A., Matis, M., Cho, B., Axelrod, J. D. (2014) Prickle/spiny-legs isoforms control the polarity of the apical microtubule network in planar cell polarity. Development 141: 2866-2874. PubMed ID: 25005476

Papayannopoulos, V., et al. (1998). Dorsal-ventral signaling in the Drosophila eye. Science 281(5385): 2031-4. PubMed Citation: 9748163

Rauskolb, C. and Irvine, K. D. (1999). Notch-mediated segmentation and growth control of the Drosophila leg. Dev. Biol. 210(2): 339-50. PubMed Citation: 10357895

Rogulja, D., Rauskolb, C. and Irvine, K. D. (2008). Morphogen control of wing growth through the fat signaling pathway. Dev. Cell 15: 309-321. PubMed Citation: 18694569

Simon, M. A. (2004). Planar cell polarity in the Drosophila eye is directed by graded Four-jointed and Dachsous expression. Development 131(24): 6175-84. 15548581

Strutt, H. and Strutt, D. (2002). Nonautonomous planar polarity patterning in Drosophila: Dishevelled-independent functions of Frizzled. Dev. Cell 3: 851-863. 12479810

Strutt, H., Mundy, J., Hofstra, K. and Strutt, D. (2004). Cleavage and secretion is not required for Four-jointed function in Drosophila patterning. Development 131: 881-890. 14757640

Tokunaga, C. and Gerhart, J. C. (1976). The effect of growth and joint formation on bristle pattern in D. melanogaster. J. Exp. Zool. 198(1): 79-95. 77030532

Villano, J. L. and Katz, F. N. (1995). four-jointed is required for intermediate growth in the proximal-distal axis in Drosophila. Development 121(9): 2767-77. PubMed Citation: 7555705

Waddington, C. H. (1943). The development of some `leg genes' in Drosophila. J. Genet. 45: 29-43

Willecke, M., et al. (2008). Boundaries of Dachsous Cadherin activity modulate the Hippo signaling pathway to induce cell proliferation. Proc. Natl. Acad. Sci. 105(39): 14897-14902. PubMed Citation: 18809931

Yang, C.-h., Axelrod, J. D. and Simon, M. A. (2002). Regulation of Frizzled by Fat-like cadherins during planar polarity signaling in the Drosophila compound eye. Cell 108: 675-688. 11893338

Zeidler, M. P., Perrimon, N. and Strutt, D. I. (1999). The four-jointed gene is required in the Drosophila eye for ommatidial polarity specification. Curr. Biol. 9: 1363-1372. PubMed Citation: 10607560

Zeidler, M. P., Perrimon, N. and Strutt, D. I. (2000). Multiple Roles for four-jointed in planar polarity and limb patterning. Dev. Bio. 221: 181-196. PubMed Citation: 11112323

Biological Overview

date revised: 25 April 2019

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