InteractiveFly: GeneBrief

fat : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

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 at 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 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 to failures in cell adhesiveness and cell recognition. Double mutant combinations with alleles of other genes, e.g., of the Epidermal growth factor receptor pathway, modify ft clonal phenotypes, indicating that adhesiveness is modulated by intercellular signaling. In addition, mutant ft cells show smaller cell sizes during proliferation and abnormal cuticular differentiation; this is reflective of cell membrane and cytoskeleton anomalies, that are not modulated by the Egfr pathway (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).

The first indication that Ft functions during PCP signaling in the eye came from examining flies homozygous for the weak, viable ft allele, ft¹. A small fraction of ommatidia with reversed dorsal-ventral (d-v) polarity were consistently observed. To examine the effects of stronger ft alleles on ommatidial polarity, FRT-mediated mitotic recombination was used to generate clones of cells homozygous for the lethal ft alleles, ft^l(2)fd or ft^Gr-V. Ommatidia located within the ft mutant tissue were constructed normally, but they frequently adopted the reversed d-v polarity form (~40% for ft^l(2)fd, ~50% for ft^Gr-V). In addition to polarity reversals within the ft mutant tissues, occasional reversals of polarity in wild-type ommatidia bordering the polar side of the mutant tissue were also observed. This effect resembles the nonautonomous phenotypes previously reported for fz mutant clones in the eye (Yang, 2002).

The presence of a randomized pattern of d and v type ommatidia within ft mutant tissue suggests that Ft is required to correctly bias R3/R4 specification. To confirm that the initial pattern of R3 and R4 specification is randomized in ft mutant ommatidia, the expression pattern of an R4-specific marker, E(spl)mdelta0.5, was examined in ft^l(2)fd and ft^Gr-V clones. This marker consists of a portion of the enhancer region of the E(spl) gene, a transcriptional target of N activation, fused to a ß-galactosidase (ß-gal) reporter. In wild-type ommatidia, this marker is initially expressed in both R3/R4 precursor cells, but then becomes stronger in the polar cell since this cell is specified as R4. Within the ft ommatidial clusters, the R4-specific marker is still strongly expressed in only one member of the R3/R4 pair. However, the cell expressing the R4 marker frequently occupies the position normally taken by the equatorial cell, indicating that the pattern of R3/R4 cell fate specification is reversed (Yang, 2002).

Because the presence of higher Fz signaling in the equatorial member of the R3/R4 precursor pair is a crucial determinant of R3/R4 specification, these results suggest that Ft may function in the placement or interpretation of positional cues controlling the equatorial/polar bias of Fz signaling. This model predicts that while Fz signaling should still occur in the absence of Ft, the pattern of Fz activation within an R3/R4 pair should be randomized with respect to the equator. Consistent with this prediction, two important differences between the ft and fz mutant phenotypes indicate that Fz signaling remains intact in ft ommatidia. (1) Previous studies have shown that many fz ommatidia are incorrectly formed and fail to have distinctly specified R3 and R4 cells. This phenotype indicates that the processes preventing both R3 and R4 precursor cells from adopting the same fate depend on Fz function and are inefficient when only Dl/N-mediated lateral inhibition is used to specify R3 and R4. (2) fz ommatidia frequently either fail to rotate or rotate incorrectly even when R3 and R4 cells are specified. In contrast, most ft ommatida contain uniquely specified R3 and R4 cells and rotate in the proper direction for their pattern of R3/R4 specification. These differences between the fz and ft phenotypes suggest that Fz signaling remains functional in the absence of Ft function (Yang, 2002).

To show that the absence of Ft function causes Fz signaling to occur in a randomly biased pattern within R3/R4 precursor pairs, eye discs containing ft mutant clones were immunostained for Flamingo (Fmi)/Starry Night (Stan) protein. Previous work has suggested that Fmi and Fz function together in a signaling complex at the proximal/distal (p/d) junctions between wing cells. Furthermore, the accumulation of Fmi at a p/d cell-cell boundary depends on the presence of differences in Fz signaling levels between the two cells. To first show that Fmi functions during PCP signaling in the eye, clones of cells homozygous for a strong loss-of-function fmi allele (fmi^E59) were examined. Dramatic polarity defects were found that resembled those seen in fz ommatidia. The defects included aberrant rotation and a lack of distinct R3/R4 fates (Yang, 2002).

Immunostaining eye imaginal discs for Fmi has revealed a dynamic pattern of localization within developing ommatidia. In young clusters prior to ommatidial rotation, Fmi is localized to the cell membranes of both of the R3/R4 precursors. However, as the clusters initiate rotation, Fmi shows asymmetric localization and is eventually concentrated at the cell-cell junction between the R3 and the R4 precursors and at the polar border of the R4 precursor, where R4 abuts a cell of undetermined fate. To confirm that this asymmetric localization depends on Fz signaling, Fmi localization was examined in eye discs from fz mutant animals (fz^H51/fz^KD4a). In the absence of functional Fz, Fmi was uniformly localized to the cell membranes of R3/R4 precursors without obvious signs of asymmetry. Further support for the use of asymmetric Fmi localization as a marker for Fz signaling came from observations that GFP-tagged forms of Fz and Dsh, whose localization to the p/d boundaries in wing cells are also Fz dependent and coincide with that of Fmi, similarly colocalize with Fmi within ommatidial clusters. Having established that Fmi localization can be used as a marker for Fz signaling, Fmi localization was examined in ft clones. Asymmetric localization of Fmi within individual clusters is readily apparent in ft tissue. However, the pattern of asymmetric localization is often reversed and correlates with the pattern of R3/R4 specification as shown by the direction of rotation of those clusters (Yang, 2002).

Intense Fmi accumulation is often apparent anterior to the morphogenetic furrow along the ft clonal border. Since differences in Fz levels between adjacent wing cells can recruit Fmi to cell-cell boundaries, this result suggests that differences in the levels of Ft function between adjacent cells can result in differences in their levels of Fz signaling. Thus, these experiments raise the possibility that asymmetric Fz signaling between the R3/R4 precursor cells occurs because one precursor cell has more Ft function than does its neighbor. For example, Ft function might be consistently higher in the equatorial precursor cell and act to either enhance Fz signaling in that cell or to inhibit Fz signaling in the neighboring polar precursor cell. This model makes two predictions. (1) Removal of Ft function only from the precursor cell that normally has higher Ft function should lead to reversal, rather than randomization, of the pattern of R3/R4 specification. In contrast, removal of Ft from the precursor with lower Ft function should have little effect on R3/R4 specification. (2) The ability of differences in Ft signaling within a precursor pair to determine the pattern of R3/R4 specification should depend on the presence of Fz activity (Yang, 2002).

The effect of removing Ft from one member of an R3/R4 precursor pair was assayed using clones of homozygous ft cells generated in a heterozygous background. Because there are no strict lineage relationships among ommatidial cells, the ommatidia along the clonal border were composed of random combinations of ft^-/+ and ft^-/- cells. Among these combinations were mosaic ommatidia in which only one member of the R3/R4 precursor pair possessed functional Ft. Because ft ommatidia rotate in the direction corresponding to their pattern of R3/R4 specification, the fate of the equatorial and the polar precursors in such R3/R4 mosaic ommatidia could be determined. In ommatidia that adopted the correct polarity for their position in the eye, R3 and R4 were derived from the equatorial and polar precursor cells, respectively, while in reversed polarity ommatidia, the R3 was derived from the polar precursor cell and R4 from the equatorial cell. Analysis of 166 R3/R4 mosaic ommatidia revealed that in all 71 examples where Ft function was absent from the polar R3/4 precursor cell, the equatorial cell became R3 and the resulting ommatidia displayed normal polarity. In contrast, loss of Ft function from the equatorial cell resulted in the polar cell becoming R3 and a reversal of ommatidial polarity in 88 of 95 examples. These results demonstrate that Ft normally acts in the equatorial cell to promote the correct pattern of R3/R4 specification. These data also indicate that in ommatidia lacking Ft function only in the equatorial cell, the Ft present in the polar cell actively directs the reversal of the pattern of R3/R4 specification. This is apparent in the observation that removal of Ft function from only the equatorial cell usually reverses the R3/R4 specification pattern. In contrast, loss of Ft function from both precursor cells leads to a random pattern of R3/R4 specification. The ability of strong differences in Ft function to override the normal positional cues instructing R3/R4 specification is consistent with the idea that the normal equatorial/polar bias in Fz signaling results at least in part from the presence of higher Ft function in the equatorial precursor cell (Yang, 2002).

To demonstrate that Fz is required for differences in Ft levels between the R3/R4 precursor cells to dictate the pattern of R3/R4 specification, ft clones were generated in the eyes of fz^KD4a/fz^H51 animals. Analysis of 23 R3/R4 ft mosaic ommatidia has revealed that differences in Ft function failed to determine the pattern of R3/R4 development in the absence of Fz. ft^-/- R3/R4 precursor cells in the fz background adopted either the R3 or R4 fate with roughly equal frequency (39% specified as R4, 61% as R3), in marked contrast to ft^-/- cells in mosaic ommatidia generated in a fz⁺ background (95% specified as R4, 5% as R3) (Yang, 2002).

These data support a model in which a higher Fz signaling in the equatorial member of each R3/R4 precursor pair results from the presence of higher Ft function in that cell. The next experiments sought to discover the mechanisms that control the level of Ft function within each R3/R4 precursor pair. One possibility is that Ft could be expressed in a graded fashion from the equator toward the poles. To investigate this possibility, antibodies were raised against the intracellular domain of Ft and wild-type eye discs were stained. It was found that Ft is not expressed in a detectable graded fashion. Instead, Ft appears to be uniformly distributed along most cell membranes and overlaps with Dsh-GFP prior to the asymmetric localization of Dsh-GFP in the region near the morphogenetic furrow where R3/R4 cell fate decisions are being made (Yang, 2002).

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

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

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

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

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

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

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

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 (fj^N7) 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 ds^UA071 flies. Since the lack of a recognizable equator in homozygous ds mutant eyes made it impossible to designate normal versus reversed polarity ommatidia in fj^N7 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 ds^UA071 clones in either wild-type or homozygous fj^N7 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 arm^H8.6 mutation. ds-lacZ expression was severely reduced in these clones. Combined with previous studies of fj-lacZ expression, these data suggest that the ds and fj expression gradients result in large part from the presence of a gradient of Wg signaling that increasingly activates ds and inhibits fj expression near the poles. It is worth emphasizing that the receptor mediating the effects of Wg on fj and ds expression is likely to be another member of the Fz family, perhaps dFrizzled2 (dFz2), rather than Fz itself. This is evident from the observation that fj-lacZ expression is not affected by the loss of Fz function (Yang, 2002).

Genetic epistasis experiments strongly suggest that Fj, Ds, and Ft act together to control PCP signaling. Further support for their placement in a common pathway came from an analysis of fj-lacZ expression. Fj can regulate its own expression. fj overexpression in eye disc clones represses fj-lacZ expression within the clones. When fj-lacZ expression is examined in ft and ds eye disc clones, the loss of Ft leads to an increase in fj-lacZ reporter expression, while the loss of Ds leads to a reduction in fj-lacZ expression. These results suggest that Fj regulates its own expression via a pathway involving Ft and Ds. While the importance to ommatidial polarity of the fj transcriptional regulation by Fj, Ft, and Ds is presently unclear, these data provide further evidence that these proteins can act in a common signaling pathway (Yang, 2002).

Planar polarization in the Drosophila eye is evident in the consistent orientation of the ommatidial units with respect to the equator and poles of the eye. The key step in this process is the establishment of a higher level of Fz PCP signaling activity in the equatorial member of the R3/R4 precursor pair. This data is relevent to understanding the mechanisms by which the R3/R4 precursor cells within an ommatidium sense which cell is closer to the equator. It has been shown that Ft and Ds, play essential roles in this process. Support for this conclusion comes from analyzing ommatidial development in the absence of Ft or Ds activity. In either case, the orientation of ommatidial polarity and Fz-dependent signaling events are randomized with respect to the equator. From this data, a model is proposed for the establishment of PCP in which the presence of higher Ft function in the equatorial cell allows it to attain a higher level of Fz signaling than its polar neighbor. This idea is supported by analysis of ommatidia in which one member of the R3/R4 precursor pair lacks Ft function. This mosaic analysis has shown that Ft normally functions from the equatorial precursor cell (the R3) to promote correct ommatidial polarity and that experimental alteration resulting in the absence of Ft from the equatorial precursor cell allows Ft present in the polar cell to direct the polar cell to become an ectopic R3 (Yang, 2002).

A second feature of the proposed model is that greater Ft function in the equatorial cell results from the presence of greater Ds function in the adjacent polar precursor cell. Important support for this idea comes from mosaic experiments showing that Ds acts primarily in the polar precursor cell and from genetic epistasis experiments between ft and ds that are consistent with Ds acting through the modulation of Ft. The final element of the model is that the presence of greater Ds function in the polar precursor cell results from two sources. ds transcription is graded (high at the poles, low at the equator) across the eye disc due to a stimulation of ds transcription by the secreted ligand Wg, which is expressed at the poles. A second source of Ds regulation appears to come from the action of Fj, which is expressed in a graded fashion from the equator and acts in the equatorial cell, in which it is more highly expressed, to promote R3 fate. Importantly, results from genetic epistasis experiments between fj and ds are consistent with Fj regulating ommatidial polarity by modulating Ds function (Yang, 2002).

While the proposed model provides a genetic framework for an understanding of how Ft, Ds, and Fj collaborate to regulate Fz signaling and the orientation of planar polarity, many issues remain that will only be answered by biochemical analysis of the signaling pathway. Among the most important of these is whether the regulation at each step of the pathway occurs in a cell-autonomous or in a nonautonomous 'crosstalk' manner. Because the specification of the R3 and R4 cells is determined by a competition between them for the higher Fz signaling state, two possible mechanisms exist for each step in the proposed pathway. Using the regulation of Ft by Ds as an example, the presence of higher Ds in the polar cell may act in a cell-autonomous manner to inhibit Ft function, causing the polar cell to have less Ft activity than its equatorial neighbor. Alternatively, Ds in the polar cell could promote Ft function in the neighboring equatorial cell, perhaps as a result of cadherin domain interactions between Ds and Ft across the junction of the two cells. It is worth noting that cadherin domain interactions of Ft or Ds with Fmi may also play an important role in Fz regulation. For example, the ability of Fmi to inhibit Fz signaling in the wing raises the possibility that Ft on the surface of the equatorial cell might bind to and stimulate the activity of Fmi in the neighboring polar cell. Addressing these different mechanisms of action will require detailed understanding of the architecture of the cell-cell junction formed between the R3/R4 precursor cells (Yang, 2002 and references therein).

One important implication of this study regards the role of Wnt proteins in regulating PCP. While the central role of Fz in PCP signaling has led to the proposal that a Wnt gradient might directly control the orientation of planar polarization by acting as a graded Fz ligand, appropriately expressed Wnts have not been reported. This analysis provides a possible explanation for the directional bias of Fz signaling that does not rely directly on a Wnt molecule to provide a graded Fz activating signal. It is important to note, however, that these experiments do not rule out the possibility that a Wnt molecule might participate in planar polarity signaling in the eye by providing a basal level of Fz activation that is then modified by the action of Fj, Ds, and Ft (Yang, 2002).

Thus a model has been provided for the directional biasing of Fz signaling in the Drosophila eye. These data illustrate how a diffusible morphogen, Wg in this case, can direct PCP by establishing opposing gradients of ds and fj expression that then regulate Ft and Fz. This analysis raises the possibility that the core of this proposed mechanism, the ability of opposing gradients of ds and fj expression to directionally bias Ft and Fz signaling, might comprise an evolutionarily conserved cassette that is deployed, under the control of different morphogens, to orient PCP signaling in other tissues. Several reports support this idea. (1) recent work has shown that Fj regulates PCP signaling in the Drosophila wing and is expressed in a gradient along the p/d axis; (2) it has been shown that Ds also regulates PCP in the wing and is expressed in a pattern complementary to that of Fj. Together, these results suggest that Ds and Fj collaborate to regulate planar polarity signaling in Drosophila tissues other than the eye. (3) It has been suggested that mice affected by the Ames waltzer hearing loss syndrome, which results from mutations in the cadherin superfamily member Pch15 and is characterized by extensive disorganization and degeneration of the cochlear and vestibular neuroepithelium, have early defects in the orientation of their outer hair cells (Alagramam, 2001; Raphael, 2001). This early phenotype may represent a defect in the establishment of PCP and thus suggest a conserved role in PCP signaling for other nonclassical cadherins besides Ft and Ds (Yang, 2002 and references therein).

The cadherins fat and dachsous regulate dorsal/ventral signaling in the Drosophila eye

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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).

Fat-regulated adaptor protein Dlish binds the growth suppressor Expanded and controls its stability and ubiquitination

The Drosophila protocadherin Fat controls organ size through the Hippo pathway, but the biochemical links to the Hippo pathway components are still poorly defined. Previous work has identified Dlish, an SH3 domain protein that physically interacts with Fat and the type XX myosin Dachs, and has shown that Fat's regulation of Dlish levels and activity helps limit Dachs-mediated inhibition of Hippo pathway activity. This study characterizes a parallel growth control pathway downstream of Fat and Dlish. Using immunoprecipitation and mass spectrometry to search for Dlish partners, Dlish was found to binds the FERM domain growth repressor Expanded (Ex); Dlish SH3 domains directly bind sites in the Ex C terminus. It was further shown that, in vivo, Dlish reduces the subapical accumulation of Ex, and that loss of Dlish blocks the destabilization of Ex caused by loss of Fat. Moreover, Dlish can bind the F-box E3 ubiquitin ligase Slimb and promote Slimb-mediated ubiquitination of Expanded in vitro. Both the in vitro and in vivo effects of Dlish on Ex require Slimb, strongly suggesting that Dlish destabilizes Ex by helping recruit Slimb-containing E3 ubiquitin ligase complexes to Ex (Wang, 2019).

The intracellular domain (ICD) of the giant Drosophila protocadherin Fat reduces cell proliferation in imaginal disc tissues by regulating the Hippo pathway, an effect potentiated by heterophilic binding between Fat and the protocadherin Dachsous (Ds). The Fat ICD increases the activity of NDR family kinase Warts, the Drosophila homolog of vertebrate LATS and the final effector kinase in the Hippo pathway, and decreases the activity of the Warts target Yorkie (Yki), the Drosophila homolog of the vertebrate YAP and TAZ transcriptional coactivators. Active Warts phosphorylates and inhibits Yki by increasing the binding between Yki and its cytoplasmic tethers. In the absence of Fat, Warts activity is reduced, increasing the proportion of Yki that enters the nucleus with its TEAD-family cofactor Scalloped (Sd), driving transcription and overgrowth of imaginal disc epithelia (Wang, 2019).

While Fat is one of the best-studied transmembrane regulators of the Hippo pathway, the biochemical pathways that link Fat's ICD to changes in Warts and Yki activity have not been fully elucidated. The portions of Fat's ICD that suppress growth lack obvious catalytic or protein-binding motifs and, until recently, binding partners. However, recent work indicates that Fat's ICD binds to the cytoplasmic SH3-domain protein Dlish (also known as Vamana), reducing Dlish levels and activity, and thereby regulating a Dlish-binding partner, the atypical type XX myosin Dachs (Zhang, 2016; Misra, 2016) (see Model of inputs from the Fat, Ds, and Crumbs ICDs into the Hippo growth control pathway). Fat, Dlish, Dachs, and Warts are all concentrated at the subapical cell cortex of disc epithelial cells near their adherens junctions, although some subapical Warts is also concentrated at nonjunction sites, and for both Dlish and Dachs this depends on the formation of a Dlish-Dachs complex. When Fat is lost, the subapical levels of Dlish and Dachs greatly increase, an effect specific to the Fat branch of the Hippo pathway. The increased Dachs binds and inhibits Warts by altering its conformation and reducing its levels, thereby inducing Yki-mediated overgrowth (Wang, 2019).

Previous evidence suggested that the increased Dlish of fat mutants stimulates growth only by increasing subapical Dachs, rather than through any direct effect on Warts or Yki. Unlike Dachs, Dlish does not bind Warts. And while Dlish is necessary and sufficient for the localization and activity of wild-type Dachs, the overgrowth induced by a membrane-targeted Dachs construct is not reduced by the loss of Dlish; concentrating Dachs at the membrane is sufficient to bypass Dlish. Instead, Dlish provides a physical link between Dachs and the Fat ICD, along with the DHHC palmitoyltransferase Approximated (App), which can bind Fat and Dachs and palmitoylate Dlish. Loss of either Dlish or App disrupts Fat's ability to regulate Dachs levels and Yki activity. In the simplest view, the Fat ICD binds and inhibits Dlish and App, reducing their ability to regulate Dachs; when Fat is lost, App and palmitoylated Dlish are free to bind Dachs and concentrate it near the subapical cell membrane where it inhibits Warts. Dachs is also needed to concentrate the Dlish/Dachs complex in the cortex, likely by binding to the actin cytoskeleton or other scaffolds; thus, the reduction of Dachs by the Fat-tethered ubiquitin ligase Fbxl7 provides a parallel route for regulating Dlish and Dachs activity. The Ds ICD also binds and recruits Dachs and Dlish, but it is uncertain how strongly the endogenous Ds ICD affects the Hippo pathway (Wang, 2019).

While Dachs plays a major role in growth control through its ability to inhibit Warts, this study shows that this is not the sole mediator of Dlish activity. Rather, Dlish also regulates a parallel pathway mediated by the growth-inhibiting FERM domain protein Expanded (Ex). Ex regulates the Hippo pathway at multiple levels that are distinct from the Dachs-mediated alterations in Warts conformation. Ex binds Hippo, Warts, and the pathway modulators Merlin and Kibra and stimulates the phosphorylation and activity of Warts; Ex can also bypass Warts by binding and inhibiting Yki (Wang, 2019).

Indeed, Fat was originally linked to the Hippo pathway, not through Dachs, but through Ex: loss of Fat decreases Ex levels in the subapical cell cortex. The Ex decrease is particularly striking as it is at odds with the increased Yki-driven ex transcription caused by loss of Fat, indicating that the effect is posttranscriptional; the effect is also specific for the Fat branch of the Hippo pathway, as increasing Yki activity through other branches increases both ex transcription and Ex protein levels as part of a negative feedback loop (Wang, 2019).

This study shows that mimicking the effects of Fat loss by increasing Dlish levels has a growth-inducing activity that is independent of the Dachs myosin, and thus Dachs-mediated inhibition of Warts.Two of the three SH3 domains of Dlish bind directly to multiple sites in Ex, Dlish decreases Ex protein levels in wing imaginal discs independently of ex transcription, and that without Dlish the loss of Fat no longer reduces Ex protein levels. Previous studies showed that Ex levels are reduced by ubiquitination mediated by the Ex-binding F-box E3 ubiquitin ligase Slimb and the Skp-Cullin-F-box (SCF) complex, a process stimulated by the Ex-binding transmembrane protein Crumbs. This study will confirm and extend previous finding that Dlish binds to Slimb and shows that Dlish stimulates the Slimb-dependent ubiquitination of Ex in vitro (Wang, 2019).

Yorkie drives supercompetition by non-autonomous induction of autophagy via bantam microRNA in Drosophila

Mutations in the tumor-suppressor Hippo pathway lead to activation of the transcriptional coactivator Yorkie (Yki), which enhances cell proliferation autonomously and causes cell death non-autonomously. The mechanism by which Yki causes cell death in nearby wild-type cells, a phenomenon called supercompetition, and its role in tumorigenesis remained unknown. This study shows that Yki-induced supercompetition is essential for tumorigenesis and is driven by non-autonomous induction of autophagy. Clones of cells mutant for a Hippo pathway component fat activate Yki and cause autonomous tumorigenesis and non-autonomous cell death in Drosophila eye-antennal discs. This study found that mutations in autophagy-related genes or NF-κB genes in surrounding wild-type cells block both fat^-induced tumorigenesis and supercompetition. Mechanistically, fat mutant cells upregulate Yki-target microRNA bantam, which elevates protein synthesis levels via activation of TOR signaling. This induces elevation of autophagy in neighboring wild-type cells, which leads to downregulation of IκB Cactus and thus causes NF-κB-mediated induction of the cell death gene hid. Crucially, upregulation of bantam is sufficient to make cells to be supercompetitors and downregulation of endogenous bantam is sufficient for cells to become losers of cell competition. These data indicate that cells with elevated Yki-bantam signaling cause tumorigenesis by non-autonomous induction of autophagy that kills neighboring wild-type cells (Nagata, 2022).

The data reveal that the Hippo pathway mutant fat clones cause supercompetition by inducing autophagy-mediated cell death in surrounding wild-type cells via NF-κB-mediated induction of hid. The autophagy induction in wild-type cells depends on Yki-bantam-mediated activation of TOR signaling in neighboring fat mutant cells. This mechanism is similar to what was observed in the elimination of ribosomal protein or Hel25E mutant loser clones when surrounded by wild-type cells. This is particularly interesting in two ways: first, it suggests that different types of cell competition, namely elimination of unfit cells by wild-type cells and elimination of wild-type cells by supercompetitors, are driven by the common mechanism, and second, it indicates that induction of autophagy in loser cells is non-autonomous, as even wild-type cells elevate autophagy when juxtaposed to supercompetitors. Although the mechanism by which autophagy is induced in loser cells nearby winner cells remains unknown, observations in this study in conjunction with the previous data on the elimination of ribosomal protein or Hel25E mutant clones suggest the possibility that relative difference in protein synthesis levels between cells plays a critical role in autophagy induction (Nagata, 2022).

The mechanism by which elevated autophagy induces hid expression via NF-κB still remains to be elucidated. Elevated autophagy results in downregulation of IκB protein Cactus. IκB is known to be degraded by the ubiquitin-proteasome system. On the other hand, elevated autophagy by starvation or rapamycin treatment was shown to cause degradation of IκB and thus activate NF-κB in mouse fibroblast. Together, the data suggest the possibility that IκB is degraded by selective autophagy in losers of cell competition (Nagata, 2022).

The observations of this studsy intriguingly show that non-autonomous cell death in wild-type cells promotes fat-induced tumorigenesis. This supports the idea that cancer cells expand their territories within the tissue by cell competition during malignant progression of tumors. While the mechanism by which wild-type cell death fuels neighboring tumorigenesis is an important open question, it may involve compensatory proliferation triggered by mitogenic factors secreted from dying cells. Intriguingly, it has been reported in Drosophila eye-antennal discs that clones of malignant tumors caused by Ras activation and cell polarity defects induce autophagy in surrounding wild-type cells, which in this case do not cause cell death but provide nutrient such as amino acids to neighboring tumors to promote their growth. Clones of cells overexpressing activated form of Yki were also shown to induce autophagy in neighboring cells, but in this case non-autonomous autophagy does not have a role in promoting tumorigenesis. Thus, non-autonomous autophagy may have multiple roles and mechanisms in regulating tissue homeostasis and tumorigenesis (Nagata, 2022).

Given that the Hippo pathway is conserved throughout evolution and that YAP-mediated cell competition occurs in mammalian systems as well, autophagy-mediated cell death may play an important role in mammalian cell competition. Notably, in a mouse liver cancer model, hyperactivation of YAP in peritumoral hepatocytes triggers regression of primary liver tumors and melanoma-derived liver metastases. Thus, further studies on the mechanism of Hippo-signaling-mediated supercompetition in Drosophila may provide a novel therapeutic strategy against human cancers (Nagata, 2022).

REGULATION

Fat and Wingless signaling oppositely regulate epithelial cell-cell adhesion and distal wing development

Development of organ-specific size and shape demands tight coordination between tissue growth and cell-cell adhesion. Dynamic regulation of cell adhesion proteins thus plays an important role during organogenesis. In Drosophila, the homophilic cell adhesion protein DE-Cadherin regulates epithelial cell-cell adhesion at adherens junctions (AJs). This study shows that along the proximodistal (PD) axis of the developing wing epithelium, apical cell shapes and expression of DE-Cad are graded in response to Wingless, a morphogen secreted from the dorsoventral (DV) organizer in distal wing, suggesting a PD gradient of cell-cell adhesion. The Fat (Ft) tumor suppressor, by contrast, represses DE-Cad expression. In genetic tests, ft behaves as a suppressor of Wg signaling. Cytoplasmic pool of ß-catenin/Arm, the intracellular transducer of Wg signaling, is negatively correlated with the activity of Ft. Moreover, unlike that of Wg, signaling by Ft negatively regulates the expression of Distalless (Dll) and Vestigial (Vg). Finally, Ft is shown to intersect Wnt/Wg signaling, downstream of the Wg ligand. Fat and Wg signaling thus exert opposing regulation to coordinate cell-cell adhesion and patterning along the PD axis of Drosophila wing (Jaiswal, 2006).

Cells of the dorsoventral (DV) boundary in the wing imaginal disc synthesize Wg. The DV boundary marks the distal end of the growing appendage, while the future hinge region, displaying Wg expression in two concentric rings, marks the proximal wing. The lacZ reporter of the quadrant enhancer of vestigial (vg), Q-vg-lacZ marks the entire distal wing [i.e. the presumptive wing blade (pouch) (Jaiswal, 2006).

In optical sections of the imaginal disc epithelium, AJs are visualized in the XY or XZ planes based on immunolocalization of DE-Cad and ß-catenin/Arm, besides binding with fluorochrome conjugated Phalloidin to F-actin. Both ß-catenin/Arm and DE-Cad display characteristic upregulation across the DV boundary along the PD axis of the wing imaginal disc. Optical sections along the XY plane reveal higher levels of DE-Cad localization and narrower apical circumferences in the AJs of cells flanking the DV boundary when compared with those of the more proximally located cells. Optical sections along the XZ plane further confirmed upregulation of DE-Cad. Thus, along the PD axis of the wing disc, cell shapes and DE-Cad levels are graded (Jaiswal, 2006).

Whether the PD gradient of cell shape and DE-Cad levels are linked to Wg signaling was tested. Somatic clones displaying constitutive Wg signaling (induced by overexpression of Dsh or of a degradation resistant variant of ß-catenin/Arm, Arm^S10) induce cell-autonomous upregulation in the levels of DE-Cad and apical cell constrictions. Somatic clones expressing secreted Wg, however, are expected to induce non-cell-autonomous effects. Indeed, these clones induced non-cell autonomous and graded upregulation in the levels of DE-Cad in the AJs and changes in apical cell shapes. In the presumptive hinge region, Wg overexpression produces a more striking pattern of non-cell autonomous changes in cell shapes: cells neighboring the Wg-expressing cells appear to organize as whorls around the former and display epithelial misfolding (Jaiswal, 2006).

Furthermore, expression of GPI-anchored DFz2 receptor GPI-DFz2, which compromises Wg signaling, obliterates the characteristic PD gradient in the levels of DE-Cad and F-actin in the AJs. Finally, loss of Wg expression in the DV boundary of wing imaginal disc of N^ts mutants grown at a restricted temperature also abolishes the PD gradient of DE-Cad and apical cell shapes. To further test if apical cell constrictions are linked to elevated levels of DE-Cad in AJs, DE-Cad was expressed in somatic clones. These clones were apically constricted, consistent with the role of DE-Cad/E-Cad in remodeling cell shape and tissue architecture. These results thus link Wg signaling to the PD gradient in the levels of DE-Cad and apical cell shapes in the wing imaginal discs (Jaiswal, 2006).

Somatic clones with altered cell-cell adhesion sort out from their neighbors and display smooth clone borders. Indeed, somatic clones displaying gain of Wg signaling owing to Dsh or Arm^S10 misexpression sort out from their neighbors and display smooth clone borders, akin to those misexpressing DE-Cad. Wg signaling may alter cell-cell adhesion by enhancing recruitment of ß-catenin/Arm to the AJs and/or by its transcriptional input. In many cell types, for example, expression of cadherins rather than the levels of catenins appears to be the rate-limiting step of Catenin-Cadherin complex formation at AJs and cell-cell adhesion. Wild type ß-catenin/Arm (Arm^S2), when overexpressed, does not transduce Wg signaling. Somatic clones overexpressing Arm^S2 display 'wiggly' clone borders, unlike those expressing Dsh or Arm^S10. Thus, expression of ß-catenin/Arm alone, without a concomitant enhancement of Wg signaling, fails to alter cell-cell adhesion. Cell-cell adhesion in wing imaginal disc epithelium is therefore likely to be regulated by transcriptional input from Wg signaling (Jaiswal, 2006).

To test if canonical Wg signaling regulates DE-Cad expression, the response of its lacZ reporter, DE-Cad-lacZ, was examined. Cells receiving high threshold of Wg signaling in the wing imaginal discs, as in those flanking the DV boundary, displayed higher levels of DE-Cad-lacZ reporter activity when compared with those further away from the source of Wg expression. Furthermore, somatic clones expressing Arm^S10 or Dsh display cell-autonomous activation of the DE-Cad-lacZ. Finally, clones expressing the secreted Wg induce non-cell-autonomous activation of DE-Cad-lacZ: i.e., in cells within and surrounding the clones. Together, these results suggest that regulation of DE-Cad by the long-range activity of the Wg morphogen sets up the PD gradient of cell-cell adhesion and cell shape in the distal wing (Jaiswal, 2006).

Somatic clones lacking Ft (ft^-/ft^-), marked by loss of GFP, display overgrowth and altered cell-cell adhesion with characteristic circular and smooth clone borders, unlike the 'wiggly' borders of their wild type (ft⁺/ft⁺) twins that are marked by brighter GFP. Furthermore, cells lacking Ft displayed upregulation of DE-Cad in their AJs and DE-Cad-lacZ. By contrast, when Ft was overexpressed, levels of both DE-Cad or DE-Cad-lacZ were downregulated. Besides, following overexpression of Ft in the posterior wing compartment, cells flanking the DV boundary displayed wider apical circumferences when compared with those of the anterior wing compartment. These results suggest that Ft regulates DE-Cad expression, cell-cell adhesion and apical cell shapes in the distal wing (Jaiswal, 2006).

The results suggest that by regulating DE-Cad expression, Wg signaling integrates cell-cell adhesion with tissue growth and pattern. Regulation of DE-Cad expression could be a prevalent mechanism for coordination of the emerging pattern in an organ primordium with the spatial control of its cell-cell adhesion. For example, DE-Cad levels are also upregulated in cells flanking the stripe of cells along the AP boundary that express the morphogen Decapentaplegic (Dpp); misregulation of Dpp signaling also affects DE-Cad expression. The Ft tumor suppressor, by contrast, negatively regulates DE-Cad expression in the distal wing. This may also explain the inverse correlation between the levels of DE-Cad in AJs and the activity of Ft. Thus, besides its heterophilic binding with Ds, Ft controls cell-cell adhesions at AJs by regulating DE-Cad expression (Jaiswal, 2006).

Apart from cell-cell adhesion, DE/E-Cad regulation may impact a variety of other cellular processes and developmental mechanisms. E-Cad has been shown to mark the sites of actin assembly on cell surface. Cadherin complexes regulate cytoskeletal networks and cell polarity, while disruption of AJ associated components affects asymmetric cell division. Fat1, a mammalian homolog of Drosophila Ft, modulates actin dynamics. Interestingly, Ft also regulates orientated cell division (OCD) in imaginal epithelium, which is mirrored by orientation of the spindles of the dividing cells; OCD may also regulate organ shape along the PD axis. Misregulation of DE-Cad may thus affect the cytoskeleton and produce OCD phenotype in ft mutant discs (Jaiswal, 2006).

In both loss- and gain-of-function assays, this study shows that Ft downregulates Dll and Vg/Q-vg-lacZ in the distal wing. Although Vg/Q-vg-lacZ and Dll have not been ascertained to be the direct targets of Wg, all available evidence so far suggests that these targets positively respond to Wg signaling. These results also show that Ft and Wg signaling intersect and control distal wing growth and pattern, presumably through their opposing regulation of a common set of targets, namely, DE-Cad, Vg and Dll. Apart from Wg signaling, Dpp signaling also regulates Q-vg-lacZ; however, its long-range target, Omb is not upregulated in ft mutant clones, suggesting that regulation of distal wing targets by Ft is mediated by its intersection with Wg signaling (Jaiswal, 2006).

The results show that Ft negatively regulates Wg signaling. Loss or gain of Ft induces a telltale sign of perturbations in Wg signaling, namely, changes in the cellular pool of ß-catenin/Arm, consistent with its role as a suppressor of Wg signaling in genetic tests. The results further reveal intersection of Ft with Wg signaling downstream of the Wg ligand, while with respect to its receptor, Ft is likely to act either upstream of or parallel to Fz/Fz2. It is interesting to note here that the role of Ft in PCP regulation has also been suggested to be either parallel to or upstream of the Fz receptor. It is also noted that Ft co-localizes with neither Fz nor Fz2 and does not mediate their subcellular localization, thereby suggesting that Ft interacts with Fz indirectly. Unraveling the genetic and molecular basis of this interaction may explain how Ft straddles both the canonical (growth and cell-cell adhesion) and non-canonical (PCP) Wnt signaling pathways (Jaiswal, 2006).

One of the remarkable aspects of development of an organ primordium is that a stereotypic PCP is achieved even while it passes through dynamic changes in its size and shape. The fact that changing organ sizes/shapes does not alter PCP suggests an in-built mechanism to regulate constancy of PCP during animal development. A link between PCP and growth through the activity of Ft has been speculated, since it regulates both. Intersection of Ft and the canonical Wg signaling seen here might provide a mechanism to coordinate PCP and organ growth (Jaiswal, 2006).

Drosophila wing growth is under dynamic spatial and temporal regulation by Wg signaling. Furthermore, different thresholds of Wg signaling impact cell proliferation in their characteristic ways and activate distinct sets of PD markers. Although at a very high threshold, Wg signaling inhibits cell proliferation, at a modest threshold it has been shown to stimulate growth. It is noted that loss of Ft fails to activate Wg targets that demand a high threshold of Wg signaling, e.g., Ac, which is required for wing margin specific bristle development. Conversely, overexpression of Ft also does not lead to loss of margin bristles, suggesting that it is not a strong repressor of Wg signaling either. The short-range Wg target, fz3-lacZ, which responds to a high threshold of Wg signaling, is also not upregulated by loss of Ft. Dll responds to a higher threshold of Wg signaling than that required for Vg/Q-vg. Dll and Vg display modest and strong upregulation respectively, following loss of Ft. These results suggest that loss of Ft upregulates Wg signaling to only modest thresholds, consistent with the growth-promoting effect of the latter (Jaiswal, 2006).

Over-proliferation in ft mutant imaginal discs is induced by perturbation of as yet unidentified disc-intrinsic mechanisms that determine the discs' characteristic final sizes. The imaginal discs of ft mutants continue to grow and the extent of their over-proliferation appears to be constrained only by the developmental time available during the extended periods of their larval life. By contrast, growth in wild-type imaginal discs is determinate, which ceases after they attain their predetermined sizes even under conditions of unlimited developmental time; for example, on transplantation into wild-type adult host abdomen that can sustain development. ft mutant imaginal discs thus acquire unlimited proliferative potential, akin to immortalization, a crucial step during tumorigenesis. It is significant that the Ft tumor suppressor downregulates Wg/Wnt signaling, a pathway implicated in cancers. Several orthologs of Ft have been identified in vertebrates with diverse functions. It will thus be interesting to explore if these orthologs of Ft in higher vertebrates also interact with Wnt signaling and thereby behave as tumor suppressors (Jaiswal, 2006).

The ubiquitin ligase FbxL7 regulates the Dachsous-Fat-Dachs system in Drosophila

The atypical cadherins Dachsous (Ds) and Fat (Ft) are required to control the size and shape of tissues and organs in animals. In Drosophila, a key effector of Ds and Ft is the atypical myosin Dachs, which becomes planar polarised along the proximal-distal axis in developing epithelia to regulate tissue size via the Hippo pathway and tissue shape via modulating tension at junctions. How Ds and Ft control Dachs polarisation remains unclear. This study identified a ubiquitin ligase, FbxL7, as a novel component of the Ds-Ft-Dachs system that is required to control the level and localisation of Dachs. Loss of FbxL7 results in accumulation of Dachs, similar to loss of Ft. Overexpression of FbxL7 causes downregulation of Dachs, similar to overexpression of the Ft intracellular domain. In addition to regulating Dachs, FbxL7 also influences Ds in a similar manner. GFP-tagged FbxL7 localises to the plasma membrane in a Ft-dependent manner and is planar polarised. It is proposed that Ft recruits FbxL7 to the proximal side of the cell to help restrict Ds and Dachs to the distal side of the cell (Rodrigues-Campos, 2014).

How animal cells cooperate to build tissues of particular forms remains a fundamental unsolved problem in biology. One molecular system that controls tissue size and shape in animals is the Dachsous (Ds)-Fat (Ft) cadherin system. Ds and Ft encode large atypical cadherins that interact heterotypically to form cell-cell junctions in epithelia and are required to control tissue form in both Drosophila and mice. The Ds-Ft system is known to induce a molecular polarity in the plane of the epithelium, and this planar polarity has at least three distinct consequences, including control of tissue growth via regulation of the Hippo signalling pathway, control of tissue morphogenesis by modulating tension at cell-cell junctions, and control of the orientation of hairs, bristles and eye ommatidia in Drosophila, in part by modulating the Frizzled system of planar cell polarity (Rodrigues-Campos, 2014).

One important effector of Ds and Ft is the atypical myosin Dachs, which is thought to bind to the Ds intracellular domain and becomes planar polarised towards the distal side of each cell in the developing Drosophila wing or eye epithelium. Ds and Ft can also themselves become planar polarised, which may contribute to the polarisation of Dachs itself. Dachs then generates tension at distal cell-cell junctions to orient cell shapes, cell divisions or cell-cell rearrangements to drive tissue elongation along the proximal-distal axis of various fly epithelia. In addition, Dachs can signal to the nucleus via the Hippo pathway effector Yki (YAP/TAZ in mammals) to promote cell proliferation and tissue growth. Notably, Dachs appears to be dispensable for planar polarisation of the Frizzled system, and the ability of Ds and Ft to polarise hairs and bristles, a process that may instead depend on microtubules. This study focused on the Dachs-dependent roles of Ds and Ft in controlling tissue size and shape in Drosophila (Rodrigues-Campos, 2014).

The global cues that orient Dachs polarisation along the proximal-distal axis are known: Dachs localises distally in response to graded expression of the Ds cadherin and also of Four-jointed (Fj), a kinase that modulates Ds-Ft interactions. The gradients of Ds and Fj are opposing, such that Ds is highly expressed at the proximal end of the tissue and Fj is highly expressed at the distal end of the tissue. Yet, how epithelial cells read the slope of these gradients and translate this information into a planar polarised localisation of Dachs is still unknown. This study identified the ubiquitin ligase FbxL7 as a novel component of the Ds-Ft system that is crucial to control Dachs levels and localisation at apical cell-cell junctions (Rodrigues-Campos, 2014).

The results suggest a close relationship between the function of the Ft intracellular domain, Dco and FbxL7. Therefore whether phosphorylated Ft intracellular domain might recruit FbxL7 to the plasma membrane was tested. The localisation of GFP-tagged FbxL7 expressed in clones was examined and found that FbxL7-GFP localises to apical cell-cell junctions. By contrast, when FbxL7-GFP is expressed in ft mutant clones, it localises to the cytoplasm in a punctate pattern. A similar punctate pattern is observed when FbxL7-GFP is co-expressed with dominant-negative Dco3. Notably, the loss of Dachs that is normally induced by expression of FbxL7-GFP fails to occur when it is not recruited to the membrane by Ft and Dco. These findings support the notion that phosphorylated Ft recruits FbxL7 in order to downregulate Dachs. This model predicts that FbxL7 itself should be planar polarised to the proximal side of cells, where Ft is thought to be most concentrated and active, whereas Dachs localises to the distal side of cells away from FbxL7 and in a complex with Ds. Accordingly, low-level expression of FbxL7-GFP with ms1096.G4 reveals a planar polarised localisation, presumably to the proximal side of wing epithelial cells where Ft is known to concentrate (Rodrigues-Campos, 2014).

The above findings identify the FbxL7 ubiquitin ligase as a novel component of the Ds-Ft-Dachs system. FbxL7 is recruited to the membrane by Ft, where it then acts together with Ft and the Dco kinase to promote degradation or removal of both Dachs and Ds. The effect of FbxL7 loss and gain of function on Dachs levels are particularly strong and the phenotypic consequences in adult Drosophila closely resemble gain and loss of Dachs function, respectively. In vitro ubiquitylation assays suggest that FbxL7 can directly ubiquitylate Dachs, which is predicted to lead to its proteolytic degradation. In addition, it was observed that FbxL7 can also ubiquitylate the Ds intracellular domain in vitro and can modulate the level and localisation of Ds in vivo. It remains possible that FbxL7 acts indirectly by stabilising or activating Ft, which then acts via a different mechanism to degrade or remove Ds and Dachs proximally. The direct model is favored because of its simplicity and because ubiquitylation is generally thought to promote degradation, rather than stabilization, of proteins (Rodrigues-Campos, 2014).

These observations suggest a model in which Ft, which has been reported to localise proximally, recruits FbxL7 to the proximal side of the cell to help restrict Dachs and Ds to the distal side of the cell. These results also suggest that polarised Ds may also promote degradation or removal of Ft on the distal side so that Ft concentrates proximally, thereby assisting polar Ds-Ft bridge formation. Thus, there appears to be mutual antagonism between Ds and Ft within the same cell, as well as heterotypic Ds-Ft bridge formation between neighbouring cells, an event that then leads to loss of Dachs proximally and recruitment of Dachs distally. Such a mechanism might explain how this system can become planar polarized; however, it is still unclear how the system is able to read the slope of the Ds and Fj gradients continuously, rather than switch to a more permanently polarised state (Rodrigues-Campos, 2014).

Notably, the degree of Dachs polarisation (and the strength of its effect on Hippo signalling and tissue growth) correlates with the steepness of the Ds and Fj gradients, indicating that cells can obtain both vectorial information and a measure of steepness at the same time from the Ds-Ft system. These features of the Ds-Ft system match very well with those proposed for the hypothetical gradients originally conceived following surgical manipulation of insect development and regeneration. This identification of FbxL7 as a key player in this system will help enable further work to understand how the system can translate the steepness of the gradient into the degree of Dachs polarisation (Rodrigues-Campos, 2014).

Protein Interactions

Fat is an atypical cadherin that controls both cell growth and planar polarity. Atrophin (Grunge) is a nuclear co-repressor that is also essential for planar polarity; however, it is not known what genes Atrophin controls in planar polarity, or how Atrophin activity is regulated during the establishment of planar polarity. Atrophin is shown to bind to the cytoplasmic domain of Fat and Atrophin mutants show strong genetic interactions with fat. Both Atrophin and fat clones in the eye have non-autonomous disruptions in planar polarity that are restricted to the polar border of clones and there is rescue of planar polarity defects on the equatorial border of these clones. Both fat and Atrophin are required to control four-jointed expression. In addition mosaic analysis demonstrates an enhanced requirement for Atrophin in the R3 photoreceptor. These data suggest a model in which fat and Atrophin act twice in the determination of planar polarity in the eye: first in setting up positional information through the production of a planar polarity diffusible signal, and later in R3 fate determination (Fanto, 2003).

Mosaic analysis of ft mutant clones demonstrates a strong bias for the cell that retains ft function to become the R3 cell. This has been interpreted to indicate that Fat directly biases the cell to become an R3 cell. Atro clones also show a bias for the R3 cell to retain Atro function, supporting the model that Atro, like Fat, works in R3 fate determination (Fanto, 2003).

However, an extensive mosaic analysis found that all anterior cells (R1, R2 and R3) tend to be ft⁺, and all posterior cells (R4, R5 and R6) tend to be ft^-. It has been suggested that this bias is due to spatial considerations, cells that are polar in the precluster, undergo a 90° rotation, leaving them in a posterior position in the adult. At the polar border of ft clones, ommatidia rotate in the opposite direction to wild type, therefore the bias is reversed, leading to an increase in ft^- anterior cells. Therefore, it as been concluded that additional data are required to show that ft function is specifically needed in R3. To determine if ft and Atro are specifically required in the R3 photoreceptor, mosaic analysis of Atro, ft and wild-type clones has been undertaken. In wild-type clones, marked only by white, ommatidia at the polar border of the clone show a weak preference for posterior photoreceptors to be wild-type. This bias is strictly due to the spatial constraints of recruitment in clones. In ft clones, it has been found that at the equatorial border (where polarity is unaltered) there is no discernable difference between anterior photoreceptors subclass types; 85% of all photoreceptors that retain ft are anterior class. The increase from ~61% to 85% is probably due to the adhesive properties of ft, which result in smooth edged clones (Fanto, 2003).

By contrast, at the polar border, which is where planar polarity is altered, there is a marked tendency for ft function to be retained specifically in the R3 photoreceptor; 100% of R3 cells retained ft, whereas only 83% of all anterior photoreceptors retained ft. Mosaic analysis of Atro clones shows that the bias introduced by planar polarity (PP) alterations at the polar border of clones (similar to ft) introduces a general bias for anterior photoreceptors. However, again, the bias is stronger in the R3 cell than for other anterior photoreceptors. This increased bias in the R3 photoreceptor over the other anterior class member suggests that ft and Atro are important in R3 fate (Fanto, 2003).

The conclusion that Atro function is important for the R3 cell fate is also strongly supported by the observation that loss of Atro often results in symmetric ommatidia with two R4 cells. This is reflected by the increase in R4/R4 ommatidia seen in Atro clones in the eye disc marked by expression of the R4 marker, md-lacZ. In addition the overexpression of Atro in R3 and R4 generates symmetric ommatidia with two R3 cells. Together, these data support the proposal that Atro is needed for the R3 fate (Fanto, 2003).

The non-autonomous nature of the PP defects associated with ft and Atro mutant clones could have presented some problems to mosaic analysis. It might be expected that non-autonomous alterations in polarity would equally affect all photoreceptors, yet the data clearly show enhanced requirements for ft and Atro function in the R3 photoreceptor over other photoreceptors. In addition, the proposal that Atro is needed for the R3 cell fate is supported by analysis of the R4 marker in eye discs. Interestingly, the tendency to lose the R3 cell fate in Atro clones is seen throughout the clone, and does not appear to participate in the phenomena of equatorial rescue or polar nonautonomy (Fanto, 2003).

Because Frizzled (Fz) is also needed for R3 fate decisions, it has been suggested that Fat positively affects Fz signaling. The observation that Atro acts with Fat and also biases towards the R3 fate suggests that the regulation of Fz by Fat may not be direct. It is proposed instead that Atro is necessary for the ft-dependent bias to an R3 cell fate and for the production of a diffusible PP molecule that controls Fz activity (Fanto, 2003).

The proposal that Fat increases Fz activity, and thereby biases a cell towards the R3 fate, does not explain the non-autonomous disruptions of wild-type tissue on the polar side of ft and Atro clones, or the rescue of ft and Atro mutant tissue from wild-type tissue on the equatorial side of the clone. There are several models that could explain the non-autonomous disruptions of planar polarity. One model suggests that planar polarity is established through a 'domino effect'. This model is suggested by the striking accumulation of planar polarity components, such as Fz and Dsh on the distal edge of every cell in the wing. This observation, coupled with genetic data that suggest that high Fz activity on one side of the cell forces low Fz activity on the other side, leads to a model in which accumulation or loss of polarity in a cell leads to templating of that state onto the next cell, non-autonomously propogating PP defects. However in the eye, Fz and Dsh show differential distribution on only a subset of ommatidial precursor cells, and, importantly, intervening cells show no altered accumulation. These data argue against a simple templating model for PP in the eye (Fanto, 2003).

An alternative model suggests that the juxtapositioning of ft⁺ and ft^- tissue contributes to midline determination and emphasizes the role of Fat in inhibiting DV signaling away from the equator. This inhibition would be relieved at the equator by an unidentified molecule that would inhibit Fat function. If this model were correct, a small ft clone should mimic the situation at the equator, where Fat function is predicted to be locally inhibited. One would therefore be expected to find an ectopic equator in the middle of the clone. Instead, however, the opposite phenotype is found, since the ommatidia on the two sides of the clone point toward the middle of the clone, rather than away from it (Fanto, 2003).

The model that best explains both the equatorial rescue and polar nonautonomy of ft and Atro clones is that Fat and Atro together control expression of a planar polarity morphogen, here called 'factor X'. It is imagined that factor X is in a gradient with high levels at the equator and low levels at the poles, thus all ommatidia will appear to 'point' down this gradient. If Fat and Atro are essential to the production of factor X, then the ft/Atro mutant tissue will be void of factor X, producing a sink in the gradient. The gradient will still be pointing in the same direction initially, explaining the wild-type polarity of ommatidia at the equatorial side of the clone and 'equatorial rescue' seen in ft and Atro clones. For ommatidia at the polar edge of the clone, the gradient will be reversed, and ommatidia will point in the opposite direction. The gradient will also be disrupted outside of the clone, leading to inversions of the polarity of wild-type tissue on the polar side of the clone and 'polar nonautonomy' seen in these mutant clones. In large clones, there will be a region in the center of the clone where there is no detectable factor X, and as a result polarity will be randomized. All of these predictions are met in ft and Atro clones. Loss of Ds, which inhibits Fat function, should increase factor X. As predicted by this model, ds clones show disruptions in wild-type tissue on the equatorial side of the clone, and rescue of mutant tissue on the polar side of the clone. Without ft or ds function there would be no gradient and, consistent with this prediction, complete loss of planar polarity is seen in eyes that are homozygous for strong alleles of ft or ds (Fanto, 2003).

A gradient of Wg protein (which is high at the poles and low at the equator) initially establishes a gradient of Ds protein over the eye field. This gradient of Ds protein in turn produces a gradient of Fat activity, which, it is believed, creates a gradient of Atro activity. It is proposed that each cell will produce factor X at a level that is proportionate to the level of Atro activity, which varies according to the position of that cell in the ds and ft activity gradients. This model assumes that Factor X is a short-range diffusible molecule, which provides polarity information to ommatidial preclusters to direct their rotation. Since Fat has been shown to be upstream of Fz, it is speculated that the Atro-dependant Factor X is a ligand for Fz (Fanto, 2003).

Both ft and Atro also act in other, apparently unrelated, pathways. One of the prominent features of ft mutant larvae is the loss of growth control, which leads to dramatically overgrown discs and mutant clones that are markedly larger than their sister twin spots. However, Atro^- clones do not display overgrowth in the eye, suggesting that ft restricts growth via an Atro-independent pathway. In addition, in the adult eye Atro clones (unlike ft clones) show severe defects in photoreceptor number and type, suggesting Atro has additional roles in photoreceptor specification and/or survival that are not shared by Fat. One particularly surprising result was the finding that Atro^- clones are markedly smooth before the furrow, and that this smoothness is lost after the furrow passes. This suggests that Atro may function in a cell adhesion process that is lost upon cell differentiation (Fanto, 2003).

Dentatorubral-pallidoluysian atrophy (DRPLA) is a dominantly inherited neuronal degenerative disease characterized by the variable combination of ataxia, choreoathetosis, myoclonus, epilepsy and dementia. This disease is caused by the expansion of a polyglutamine tract within the Atrophin 1 protein. Atro is the sole fly homolog of human atrophins. Atro has been shown to act as a transcriptional co-repressor in vivo in Drosophila. Atro interacts genetically with even skipped, a transcriptional repressor, and is required for the in vivo repressive activity of even skipped. The transcriptional repressor activity of Atro has been localized to the highly conserved C-terminal region of Atro. This C-terminal region can bind to Even skipped in vitro and interacts with the minimal repression domain of Even skipped (Fanto, 2003).

This study has shown that the intracellular domain of Fat binds the C-terminal domain of Atro. The cytoplasmic expression of Atro and its interaction with Fat raises the possibility that instead of acting as a simple co-repressor, Atro functions in a more complex manner. Other transcriptional co-repressors are known to be converted to transcriptional activators upon cell signaling, and future work will determine if the interaction of Fat with Ds alters the transcriptional activity of Atro (Fanto, 2003).

Owing to the fact that Atro binds the cytoplasmic domain of Fat, a model is favored in which Atro acts downstream of Fat, possibly relaying a Fat-dependant signal to the nucleus. However, the similarity of the ft and atro loss-of-function phenotypes makes classical epistasis experiments difficult, therefore a model in which Atro acts upstream of ft cannot be excluded. Examination of the amount or subcellular distributions of Fat and Atro, suggest that Atro does not control Fat expression or localization, nor does ft control the levels or subcellular localization of Atro (Fanto, 2003).

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

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

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

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

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

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

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

The generic kinase substrates myelin basic protein and casein were not detectably phosphorylated by sFj. Thus, Fj appears to have a limited substrate specificity. Only a few proteins have been identified as being phosphorylated in the secretory pathway, and none of the responsible kinase(s) have been molecularly identified. A Golgi kinase activity, referred to as Golgi casein kinase, preferentially phosphorylates Ser or Thr residues within a S/T-X-E/D/S(Phos) consensus sequence. Because Fj does not phosphorylate casein, and the Ser residues within cadherin domains targeted by Fj do not conform to Golgi casein kinase sites, Fj is not Golgi casein kinase. Fj autophosphorylation was detected, but this reaction was weak compared to phosphorylation of Ds2-3. The autophosphorylation reaction is apparently unimolecular, because GST:Fj and sFj:V5 did not phosphorylate each other and the fraction of Fj phosphorylated was independent of concentration (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 the Ds2-3 polypeptide, but not the Ds3-4 polypeptide, even though both contain Ser²³⁶. The dicadherin constructs were based on published annotations, but in comparing Ds cadherin domains to structurally solved cadherin domains, it was realized that these misposition the intercadherin domain boundary, and consequently these constructs lacked three amino acids of the first cadherin domain. Addition of these amino acids, together with the intercadherin domain linker sequence, enabled phosphorylation of a Ds3 single-cadherin domain construct (Ishikawa, 2008).

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

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

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

Drosophila lowfat, a novel modulator of Fat signaling

The Fat-Hippo-Warts signaling network regulates both transcription and planar cell polarity. Despite its crucial importance to the normal control of growth and planar polarity, there is only a limited understanding of the mechanisms that regulate Fat. This study reports the identification of a conserved cytoplasmic protein, Lowfat (Lft), as a modulator of Fat signaling. Drosophila Lft, and its human homologs LIX1 and LIX1-like, bind to the cytoplasmic domains of the Fat ligand Dachsous, the receptor protein Fat, and its human homolog FAT4. Lft protein can localize to the sub-apical membrane in disc cells, and this membrane localization is influenced by Fat and Dachsous. Lft expression is normally upregulated along the dorsoventral boundary of the developing wing, and is responsible for elevated levels of Fat protein there. Levels of Fat and Dachsous protein are reduced in lft mutant cells, and can be increased by overexpression of Lft. lft mutant animals exhibit a wing phenotype similar to that of animals with weak alleles of fat, and lft interacts genetically with both fat and dachsous. These studies identify Lft as a novel component of the Fat signaling pathway, and the Lft-mediated elevation of Fat levels as a mechanism for modulating Fat signaling (Mao, 2009).

Recent studies have linked together the action of several tumor suppressors into a Fat-Hippo-Warts signaling network. These genes play a crucial role in growth control from Drosophila to mammals, as exemplified by the ever-increasing number of cancers that have been associated with mutations in pathway genes. Fat-Warts signaling regulates growth through a transcriptional co-activator protein, called Yorkie (Yki) in Drosophila and YAP in vertebrates. In addition, Fat influences a distinct planar cell polarity (PCP) pathway. Planar cell polarity is the polarization of cells within the plane of a tissue, and can include both polarized structures, like hairs and bristles, and polarized behaviors, such as cell division and cell intercalation (Mao, 2009).

Fat is a large member of the cadherin family, and acts as a transmembrane receptor. Fat influences the subcellular localization of both the unconventional myosin Dachs and the FERM-domain protein Expanded, and through these proteins ultimately regulates the kinase Warts. Warts then inhibits Yki by phosphorylating it: phosphorylated Yki is retained in the cytoplasm, but unphosphorylated Yki enters the nucleus to promote the transcription of target genes. The Fat PCP pathway is less well characterized, but it is partially dependent upon Dachs, and also involves Atrophin (Grunge), a transcriptional co-repressor that can bind to the Fat cytoplasmic domain (Mao, 2009).

The only Fat ligand identified is Dachsous (Ds), which like Fat is a large, atypical cadherin, and which influences the phosphorylation of Fat by Discs overgrown. ds mutants have phenotypes similar to, but weaker than, those of fat mutants, raising the possibility that there might be other ligands, or other means of regulating Fat. The Golgi kinase Four-jointed (Fj) also regulates Fat signaling, but presumably acts by modulating Fat-Ds interactions. Intriguingly, the two known Fat pathway regulators (ds and fj) are expressed in gradients in developing tissues. The vectors (directions) of these gradients parallel vectors of PCP, and experimental manipulations of ds and fj indicate that, at least in some tissues, their graded expression can direct PCP. The graded expression of ds and fj also influences the transcriptional branch of the pathway and wing growth, but in this case it is the slope rather than the vector of their gradients that appears to be instructive (Mao, 2009).

Although thus far most components of Fat signaling have been identified through genetic studies in Drosophila, protein interaction screens are an alternative approach with which to identify components of signaling pathways. A genome-wide yeast two-hybrid screen identified the product of the CG13139 gene as both a candidate Fat-interacting protein and a candidate Ds-interacting protein. This gene, which has been named lowfat (lft), encodes a small protein of unknown structure and biochemical function. It shares sequence similarity with two vertebrate genes, Limb expression 1 (Lix1) and Lix1-like (Lix1l;. Lix1 was first identified in chickens through a differential screen for genes expressed during early limb development. Subsequent analysis in mice revealed that Lix1 is actually expressed more broadly. Lix1l has been defined only by its sequence similarity to Lix1. The biological functions of these genes have not been described, although genetic mapping of a feline spinal muscular atrophy identified LIX1 as a candidate gene (Mao, 2009).

While a basic outline of Fat signaling has emerged, many steps remain poorly understood. This study shows that lft is a modulator of Fat signaling, and identified a cellular requirement for Lft in establishing normal levels of both Fat and Ds. These observations identify transcriptional regulation of lft as a potential mechanism for modulating Fat signaling through its post-translational regulation of Fat and Ds protein levels. It was also establish human LIX1L as a functional homolog of Lft, and LIX1 and LIX1L were shown to be Fat-interacting proteins, thus identifying a likely cellular function of vertebrate Lix1 genes as modulators of Fat signaling. This linkage raises the possibility that other Fat pathway components could be candidate susceptibility loci for spinal muscular atrophy (Mao, 2009).

lft mutants display decreased levels of both Fat and Ds protein staining, and presumably as a consequence exhibit a characteristic Fat pathway phenotype in the wing. In addition, lft can genetically interact with both fat and ds to cause more severe phenotypes. The lft mutant phenotype resembles weak mutant alleles of fat or ds, and lft mutants do not exhibit any additional phenotypes that could not be accounted for by effects on Fat signaling. The expression of lft itself is modulated by other signaling pathways, and differences in lft expression levels correlate with differences in Fat and Ds protein levels both in wild-type animals, and when lft levels are experimentally increased or decreased. Thus, transcriptional regulation of lft defines a mechanism for modulating Fat signaling (Mao, 2009).

Lft influences levels of both Fat and Ds. Because Fat and Ds in turn can influence levels of Lft, and because Fat and Ds also influence the localization of one another to the membrane, it is inferred that for any one of these three proteins, the influence that it has on the other two includes both direct effects, and indirect effects mediated through the third protein. In addition, the net effect observed for any one protein presumably also reflects the consequences of feedback regulation of its own levels via the other two proteins (Mao, 2009).

Given the substantial decrease in Fat staining in lft mutants, the phenotype appears surprisingly mild. This observation suggests that Fat is normally present in excess; for example, it could be that only a fraction of Fat is normally active, and that levels of Fat are not normally limiting for pathway activation. This hypothesis was supported by the observation of enhanced Fat pathway phenotypes in combination with fat¹, and would be consistent with the conclusion that Fat acts as a ligand-activated receptor, with only a fraction of Fat normally being present in the active form (Feng, 2009; Sopko, 2009). Complicating this simple explanation is the observation that the levels of the Fat ligand Ds are also reduced in lft mutants. However, because Fat signaling is influenced not only by the amount of Ds, but also by the pattern of Ds (i.e. is Ds expression graded, and how steeply), Ds can have positive or negative effects on Fat activity. Thus, it is suggested that the lft mutant phenotype might be relatively weak because decreased Fat and Ds levels, which would be expected to decrease Fat signaling, are partially offset by a flattening of the Fat and Ds expression gradients, which would be expected to increase Fat-Warts signaling (Reddy, 2008; Rogulja, 2008; Willecke, 2008; Mao, 2009 and references therein).

The observation that ds lft double mutants have more severe phenotypes than do ds or lft single mutants indicates that ds and lft can each independently influence Fat. lft and ds both influence Fat levels and localization, but even in the absence of these two genes, there was a visible difference in Fat protein staining between the wing pouch and the wing hinge. This implies that there are additional Fat regulators, and that the expression of these additional Fat regulators is differentially distributed between the wing pouch and the wing hinge. One additional Fat regulator that is differentially expressed between the pouch and the hinge is Fj, although as Fj is thought to act by influencing Fat-Ds interactions, it is not clear whether it could explain the differential Fat staining observed (Mao, 2009).

It appears that Lft is a major contributor to the normal levels of Fat. Since Lft binds to the Fat cytoplasmic domain, it presumably influences Fat protein levels through this direct binding. Different molecular mechanisms for how Lft might influence Fat (and Ds) levels can be envisioned. One attractive possibility, given that Fat and Ds are transmembrane proteins, and that Lft could co-localize with them at the sub-apical membrane, is an effect on endocytosis, but it is also possible that Lft affects them in some other way (Mao, 2009).

Because Lft is closely related to LIX1 and LIX1L, and indeed LIX1L is functionally homologous to Lft, these studies of Lft identify regulation of mammalian Fat and Ds homologs as the likely cellular functions of LIX1 and LIX1L. Consistent with this inference, these proteins could bind to the cytoplasmic domain of human FAT4, and a BLASTP search with a short sequence motif of Fat common to Ds and FAT4 (WEYLLNWGPSYENLMGVFKDIAELPD) identifies these three proteins plus the mammalian Ds homologs DCHS1 and DCHS2 as the five closest matches in protein databases. This sequence motif also exhibits weak similarity to a region of E-cadherin that has been identified as contributing to binding to β-catenin, but there is no obvious primary sequence similarity between Lft and β-catenin, and Lft did not detectably affect E-cadherin staining (Mao, 2009).

Functional studies of LIX1 and LIX1L in vertebrates have not yet been reported. However, feline LIX1 has been genetically linked to feline spinal muscular atrophy. Direct examination of human LIX1 in spinal muscular atrophy patients did not reveal any mutations. Nonetheless, the linkage of LIX1 and LIX1L to Fat signaling suggests that other members of the Fat signaling pathway should also be examined as potential candidate susceptibility loci for this debilitating disease. Murine Fat4 has been shown to be required for normal PCP in the ear and kidney; however, it is also highly expressed in the nervous system, as are murine Lix1 and Dchs genes, consistent with the expectation that these genes will interact in mammals, and might influence nervous system development (Mao, 2009).

Processing and phosphorylation of the Fat receptor

The Drosophila tumor suppressors fat and discs overgrown (dco) function within an intercellular signaling pathway that controls growth and polarity. fat encodes a transmembrane receptor, but post-translational regulation of Fat has not been described. This study shows that Fat is subject to a constitutive proteolytic processing, such that most or all cell surface Fat comprises a heterodimer of stably associated N- and C-terminal fragments. The cytoplasmic domain of Fat is phosphorylated, and this phosphorylation is promoted by the Fat ligand Dachsous. dco encodes a kinase that influences Fat signaling, and Dco is able to promote the phosphorylation of the Fat intracellular domain in cultured cells and in vivo. Evaluation of dco mutants indicates that they affect Fat's influence on growth and gene expression but not its influence on planar cell polarity. These observations identify processing and phosphorylation as post-translational modifications of Fat, correlate the phosphorylation of Fat with its activation by Dachsous in the Fat-Warts pathway, and enhance understanding of the requirement for Dco in Fat signaling (Feng, 2009).

Activation of transmembrane receptors often involves post-translational modifications, such as phosphorylation or cleavage. To investigate potential modifications, Fat was examined by Western blot analysis. In lysates of wing discs, antisera raised against the Fat intracellular domain (anti-Fat ICD) detected a prominent band with a mobility of ~95 kDa (Ft-95), and a faint band with a mobility corresponding to a much larger polypeptide (Ft-565). fat is predicted to encode a 5,147 amino acid protein, with a calculated mass of 565 kDa. Thus, Ft-95 is too small to correspond to full length Fat. Nonetheless, examination of lysates from fat mutant discs confirmed that both Ft-95 and Ft-565 are fat-dependent (Feng, 2009).

To investigate this apparent cleavage of Fat, a C-terminally tagged Fat protein (Fat:FVH) was created. When Fat:FVH was transfected into cultured Drosophila S2 cells, a band with a high apparent molecular weight, consistent with full length Fat, was observed. However, most Fat was detected in lower molecular weight bands. One correlates with the 95-kDa fragment of endogenous Fat (after accounting for the C-terminal tags), but the other appears smaller, ~70 kDa (Ft-70). Although Ft-70 was not detected when endogenous Fat was examined in imaginal discs, it could be detected in discs when Fat:FVH was overexpressed from UAS transgenes. Expression of Fat:FVH under tub-Gal4 control also confirmed that Fat:FVH is functional, because it rescued fat mutant animals. The detection of Ft-95 and Ft-70 with C-terminal epitope tags supports the conclusion that Fat is proteolytically processed. Based on their mobility, the cleavage leading to Ft-95 occurs in or near the 2 extracellular laminin G-like domains, whereas the cleavage leading to Ft-70 occurs near the transmembrane domain. A Fat construct that excludes the cadherin and EGF domains but includes most of the laminin G domain region appears to be processed to the same cleavage products as is full-length Fat, whereas a smaller Fat construct that also lacks the laminin G domains (Fat-STI-4:FVH) yields a single major band, suggesting that it is not processed (Feng, 2009).

To further characterize Fat processing, an N-terminally tagged Fat (V5:Fat) was constructed. Examination of V5:Fat by Western blotting lysates of S2 cells identified 2 bands of high apparent molecular weight, and did not detect Ft-70 or Ft-95. Although the resolving power of the gel and the lack of suitable markers precluded precise determination of the size of these large bands, their mobility is consistent with the expected detection of both full-length Fat (Ft-565) and an approximate 470-kDa N-terminal product of proteolytic processing in the Laminin G domain region (Ft-470). Double staining V5:Fat with anti-Fat ICD and anti-V5 supported the conclusion that slowest mobility isoform is full-length Fat, whereas Ft-470 lacks the Fat ICD. To characterize cleavage of V5:Fat in vivo at endogenous expression levels, the V5 tag was incorporated into a fat⁺ genomic clone, and then phiC31-mediated recombination was used to insert this into the Drosophila genome. This genomic V5:fat⁺ construct rescued fat mutants. Western blotting lysates of imaginal discs revealed that Ft-470 is more abundant than Ft-565. Because these proteins are similar in size, this differential detection is unlikely to be due to differences in blotting transfer efficiency. Hence, it is concluded that the majority of Fat protein in vivo is processed (Feng, 2009).

To investigate the nature of Fat displayed on the cell surface, biochemical experiments were performed on cultured cells. S2 cells expressing V5:Fat were incubated with anti-V5 in the absence of detergent, and then cell surface Fat bound by anti-V5 antibodies was immunoprecipitated. As a control, Fat:FVH, which includes a cytoplasmic V5 tag that should not be accessible in intact cells, was expressed. Western blot analysis of the immunoprecipitated material with anti-Fat ICD antibodies confirmed that cell surface V5:Fat is processed. In addition, these experiments demonstrate that Ft-470 and Ft-95 remain stably associated after processing. By contrast, Ft-70 was not detected, indicating that it is not associated with Ft-470. Because coimmunoprecipitation of Ft-470 and Ft-95 could be observed under reducing conditions, the association between them does not require disulfide bonds (Feng, 2009).

Because Fat processing can occur in S2 cells, which do not express detectable levels of Ds and grow as isolated cells, and processing can occur on a truncated Fat polypeptide that lacks the cadherin and EGF domains (Fat-STI:FVH), it appears that Fat processing is part of its normal maturation, rather than a regulated event. In this regard, it appears analogous to the S1 cleavage that is involved in maturation of the Notch receptor, or to the apparent processing of the Starry night/Flamingo cadherin (Feng, 2009).

Under optimal conditions, Ft-95 from wing discs runs as doublet, with a prominent lower band, a weaker upper band, and a faint smear in between. Treatment of lysates with calf intestinal alkaline phosphatase (CIP) resulted in a single sharp band ~95 kDa, with a mobility similar to the fastest of the 95-kDa mobility isoforms in untreated samples. Thus, a fraction of Ft-95 in vivo is phosphorylated. Because Ft-95 is too C-terminal to include the cadherin domains, the phosphorylation detected presumably reflects a phosphorylation of the intracellular domain, rather than Fj-mediated phosphorylation of cadherin domains. To investigate the relationship between Ft-95 phosphorylation and Fat signaling, Fat was examined in lysates of wing imaginal discs in which its putative ligand, ds, was either mutant or overexpressed. Proteolytic processing of Fat was not Ds-dependent, because Ft-95 was observed at similar levels in all cases. Mutation of ds results in enlarged wings and wing discs, and lower levels of Wts protein, a phenotype similar to, although weaker than, that of fat. Western blot analysis of Fat from ds mutant wing discs revealed that levels of the faster mobility Ft-95 band are elevated, whereas the slower mobility band (Ft-95-P) is reduced. Ds overexpression reduces wing size. When Ds was overexpressed under tub-Gal4 control, quantitative Western blot analysis of wing disc lysates identified an average increase in Ds levels of 10-fold. Strikingly, this overexpression of Ds increased the relative amount of Ft-95-P. These observations imply that the presence or absence of Ds modulates Fat phosphorylation. This was confirmed by the observation that phosphatase treatment of lysates from Ds-expressing discs collapsed the Ft-95 doublets into a single band. The visual impression that the presence of the slower mobility (Ft-95-P) isoform(s) was promoted by Ds was confirmed by quantitative line scanning of Western blot analyses (Feng, 2009).

Both mutation of fj and fj overexpression are associated with modest reductions in wing and leg size. When fj was overexpressed under tub-Gal4 control, quantitative Western blot analysis of wing disc lysates identified an average increase in Fj levels of 100-fold. This overexpression of fj was associated with an increase in the relative amount of phosphorylated Fat, and when coexpressed with ds, the increase in phosphorylated Fat appeared even greater, consistent with the reductions in wing size. Mutation of fj had only subtle affects (Feng, 2009).

Altogether, these observations identify a correlation between the presence of the Fat ligand Ds, the level of signaling through Fat to regulate Warts levels and wing growth, and the phosphorylation of the Fat cytoplasmic domain. Thus, they suggest that activation of Fat by its ligand Ds is associated with Fat phosphorylation. From the relative levels of different mobility isoforms if is inferred that in the absence of Ds overexpression, a majority of Fat is in a hypophosphorylated form, whereas overexpression of Ds promotes the production of a hyperphosphorylated form. This identification of a posttranslational modification of Fat that is promoted by Ds is consistent with the hypothesis that Fat and Ds act as receptor and ligand in a signal transduction pathway, and identifies a molecular process that appears correlated with Fat activation. Constructs that lack most of the extracellular domain, and presumably can not interact with Ds, can rescue fat mutants. However, this rescue is only partial, and has only been observed when intracellular domain constructs are overexpressed. One possibility is that interaction with ligand triggers clustering of Fat, and that overexpression of the intracellular domain allows ligand-independent clustering. This could be analogous to other signaling pathways (e.g., TGF-β, receptor tyrosine kinase), in which ligand-mediated clustering promotes phosphorylation of the cytoplasmic domain of the receptor, and for which the requirement for ligand can sometimes be bypassed by receptor overexpression (Feng, 2009).

In considering kinases that might contribute to the Ds-promoted phosphorylation of Fat, the CKIδ/ε family member Dco was a logical candidate. Genetic epistasis tests positioned dco within the Fat pathway, upstream of dachs. At the same time, dco³ exerts cell-autonomous affects on the expression of Fat target genes, which implies that it acts within receiving cells. These observations suggested Dachs or Fat as potential substrates. Initial assessment of the ability of Dco to phosphorylate them was conducted by assaying for mobility shifts in S2 cells. Dco had no effect on Dachs. By contrast, when Dco was cotransfected together with Fat, a shift in the mobility of the C-terminal cleavage products was observed. A Dco-dependent mobility shift was also observed for both the Fat-STI:FVH and Fat-STI-4:FVH constructs. Confirmation that this mobility shift was due to phosphorylation of Fat was provided by the observation that it could be reversed by phosphatase. Overexpression of a Dco construct under UAS-Gal4 control could also increase phosphorylation of endogenous Fat in vivo (Feng, 2009).

If phosphorylation of Fat by Dco is relevant to the participation of Dco in Fat signaling, then the dco³ mutation, which causes loss of Fat signaling, should impair Fat phosphorylation. Sequencing of dco³ identified 2 distinct amino acid substitutions; these were introduced into a Dco:V5 expression construct. Dco³:V5 resulted in much less shift in the mobility of Fat in S2 cells than did wild-type Dco:V5. Thus, the same amino acid changes that cause overgrowth in vivo impair Dco-dependent phosphorylation of Fat in cultured cells. To investigate whether endogenous phosphorylation of Fat could also be influenced by mutation of dco, the mobility of Fat was examined in lysates from dco³ mutant wing discs. Unphosphorylated Fat (Ft-95) appeared slightly elevated, and a distinct Ft-95-P band was no longer visible, but rather a faint smear was detected. This change in Fat mobility was confirmed by line scanning. Thus, dco³ reduces levels of phosphorylated Fat in vivo (Feng, 2009).

To explore the relationship between the Ds-promoted phosphorylation of Fat, and the Dco-dependent phosphorylation of Fat, the mobility of Fat isolated from discs simultaneously overexpressing Ds and mutant for dco³ was examined. Direct examination of Western blots, as well as line scanning, revealed that Fat mobility in these lysates was similar to that in dco³ mutants. Thus, Ds-mediated phosphorylation can be influenced by Dco. dco³ mutant clones have no obvious effect on Fat protein staining in wing imaginal discs, suggesting that they do not affect its overall levels or distribution. Nor did dco³ noticeably affect processing of Fat (Feng, 2009).

The simplest explanation for Dco-promoted Fat phosphorylation, and for dco-dependent effects on Fat signaling, would be that Dco directly phosphorylates Fat. A purified mammalian homologue of Dco (CKIδ) phosphorylated the Fat intracellular domain in vitro, but with reduced specificity, because even greater mobility shifts than those observed in vivo could be induced. CKI's are Ser/Thr kinases, and the 538 amino acid Fat ICD includes 109 Ser or Thr residues. Three different kinase site prediction programs individually predict 7, 15, or 36 CKI sites, and cumulatively identify 46 potential CKI sites. This variation emphasizes the limited accuracy of kinase site predictions. It is also noted that distinct CKI sites could act redundantly, and that among the many potential CKI sites within the Fat ICD, phosphorylation sites responsible for the evident mobility shift on SDS-PAGE gels could be distinct from sites responsible for the influence of ds or dco³ on Fat activity. Thus, the identification of specific phosphorylation sites within the Fat ICD that are required for its biological activity will ultimately be essential for confirming the importance of Dco- and Ds-promoted phosphorylation of Fat to Fat signaling (Feng, 2009).

In contrast to the overgrowth associated with dco³ mutants, dco null mutants lack discs, and dco null mutant clones grow poorly. This could reflect the participation of dco in other processes. However, targets of Fat signaling, including Wingless (WG) in the proximal wing, and Diap1, are up-regulated in dco³ mutant clones, but not in dco null (dco^le88) mutant clones. The apparent absence of fat phenotypes in dco null alleles suggests that dco³ is an unusual allele (Feng, 2009).

Dco is also known as double time, because viable alleles were independently isolated as circadian rhythm mutants. This circadian phenotype reflects a role for Dco in phosphorylating, and thereby promoting the turnover, of the circadian protein Period. This activity of Dco can be reproduced in S2 cells. Notably, Dco³:V5 was as effective as wild-type Dco:V5 at promoting Period turnover in S2 cells, whereas a circadian rhythm mutant isoform, Dco^Dbt-AR, was less effective. Thus, dco³ is impaired in promoting Fat phosphorylation, but active on another substrate (Feng, 2009).

Analysis of the Dco-Period interaction revealed that Dco and Period can be stably associated, as assayed by their ability to be coprecipitated from cultured cells. Similarly, Dco and the Fat-ICD can be coprecipitated, and this association was not impaired by the Dco³ mutations. Because Dco³ can associate with Fat, but does not efficiently phosphorylate it, Dco³ might act as an antimorphic (dominant-negative) protein by competing with wild-type kinase. Indeed, although dco³ is recessive at endogenous expression levels, when dco³ was overexpressed, aspects of the dco³ phenotype, including wing overgrowth and the induction of a Fat pathway target gene could be reproduced. By contrast, overexpression of wild-type forms of Dco does not cause detectable overgrowth phenotypes. Instead overexpression of Dco modestly decreased wing growth and slightly reduced transcription of diap1, suggesting that Fat pathway activity might be increased (Feng, 2009).

In addition to having a CKIδ/ε homologue, Drosophila also have a CKIα homologue, and in some contexts they can act partially redundantly. A partial shift in Fat ICD mobility could be detected when CKIα was expressed in S2 cells or in wing discs. Thus, CKIα can promote phosphorylation of Fat, although it appears less effective than Dco. This observation, together with the dco³ phenotypes observed when Dco³ is overexpressed, and the observation that although dco³ is defective in Fat phosphorylation, dco null mutant cells do not appear to be impaired for Fat signaling, suggest that dco³ might act as an antimorphic, or dominant negative, mutation, failing to effectively phosphorylate Fat and at the same time interfering with an ability of CKIα to phosphorylate Fat. By contrast, it is hypothesized that in dco-null mutant cells, CKIα or other kinases could phosphorylate Fat without interference. Although dco³ could not be rescued with a UAS-CKIα transgene, different CKI transgenes are inserted in different chromosomal locations, and their specific activities on Fat might be distinct. Thus, it remains possible that Dco and CKIα could be partially redundantly for Fat signaling (Feng, 2009).

Dco also participates in other pathways and processes. To determine whether the tumor suppressor phenotype of dco³ can be accounted for solely by its influence on Fat signaling, advantage was taken of the observation that overexpression of Wts under the control of a heterologous promoter (tub-Gal4 UAS-Myc:Wts) could rescue the lethality and tumor suppressor phenotype of fat mutants. The lethality and overgrowth phenotypes of dco³ were also rescued by Wts overexpression (tub-Gal4 UAS-Myc:Wts), resulting in animals that, aside from some mild wing vein phenotypes, are indistinguishable from wild-type animals overexpressing Wts. Because they are rescued simply by elevating Wts expression, dco³ mutant animals are specifically defective in Fat signaling; other essential processes that Dco participates in are not impaired (Feng, 2009).

Although Wts overexpression rescued the overgrowth and lethality of fat mutants, these animals have obvious PCP phenotypes in multiple tissues, consistent with the conclusion that Wts functions specifically in a Fat tumor suppressor pathway, and not in a Fat PCP pathway. By contrast, Wts-rescued dco³ mutants appear to have normal PCP. The absence of an obvious PCP phenotype also indicates that the influence of Dco and CKIα on PCP through phosphorylation of Dishevelled is not affected by dco³ (Feng, 2009).

To confirm the lack of influence of dco³ on PCP, dco³ mutant clones were examined. fat mutant clones in the abdomen exhibit obvious disruptions in the normal posterior orientation of hairs and bristles, but dco³ mutant clones had no effect. In addition to affecting the canonical PCP pathway, studies of the relationship between Fat and its downstream effector Dachs revealed a form of PCP in which Fat signaling causes a polarized distribution of Dachs, which can be visualized by mosaic expression of a tagged form of Dachs, Dachs:V5. In the developing wing, Dachs:V5 is present on distal cell membranes, but not on proximal cell membranes. In clones of cells mutant for fat, Dachs:V5 is equally distributed on proximal and distal membranes. In clones of cells mutants for dco³, Dachs:V5 localization is still polarized. Thus, the regulation of Dachs localization by Fat does not appear to be affected by dco³, although a weak effect on Dachs localization cannot be excluded. The absence of visible Dachs relocalization in dco³ clones appears to conflict with the hypothesis that the influence of Fat signaling on Warts depends on its ability to polarize Dachs, and further studies will be required to resolve this (Feng, 2009).

The atypical cadherin Fat is a transmembrane receptor for pathways that control PCP and transcription. This study has identified 2 posttranslational modifications of Fat. First, Fat is proteolytically processed, resulting in the production of stably associated N- and C-terminal polypeptides. The functional significance of this processing is not known, but its discovery is a necessary precursor to further experiments aimed at this question. Processing appears to be constitutive rather than regulated. Nonetheless, processing may facilitate subsequent events that regulate Fat (Feng, 2009).

Phosphorylation of the Fat cytoplasmic domain was also discovered. Phosphorylation is promoted by the Fat ligand Ds, is influenced by the Fat pathway kinase Dco, and correlates with Fat pathway activity in ds or dco³ mutant animals, or when Ds or Fj are overexpressed. These observations suggest that phosphorylation of Fat is a key step in Fat receptor activation. When Dco or CKIα are overexpressed, the phenotypic effects appear mild compared with the evident increase in phosphorylation. However, because there could be multiple CKI sites within the Fat ICD, it is possible that the phosphorylation-dependent mobility shift of Fat is a general marker of the extent of Fat phosphorylation, rather than a precise marker of phosphorylation at a site or sites required for Fat activity. Nonetheless, the observation that dco³ can be completely rescued by Warts overexpression, together with the epistasis of dachs to dco³, indicates that the tumor suppressor phenotype of dco³ is due to an impairment of Fat-Warts signaling, which occurs at or upstream of the action of Dachs. Altogether, these observations implicate Fat as the likely target of Dco activity in the Fat pathway (Feng, 2009).

Two frizzled planar cell polarity signals in the Drosophila wing are differentially organized by the Fat/Dachsous pathway

The regular array of distally pointing hairs on the mature Drosophila wing is evidence for the fine control of Planar Cell Polarity (PCP) during wing development. Normal wing PCP requires both the Frizzled (Fz) PCP pathway and the Fat/Dachsous (Ft/Ds) pathway, although the functional relationship between these pathways remains under debate. There is strong evidence that the Fz PCP pathway signals twice during wing development, and a Bidirectional-Biphasic Fz PCP signaling model has been presented which proposes that the Early and Late Fz PCP signals are in different directions and employ different isoforms of the Prickle protein. The goal of this study was to investigate the role of the Ft/Ds pathway in the context of the Fz PCP signaling model. The results lead to the following conclusions: (1) The Early Fz PCP signals are in opposing directions in the anterior and posterior wing and converge precisely at the site of the L3 wing vein. (2) Increased or decreased expression of Ft/Ds pathway genes can alter the direction of the Early Fz PCP signal without affecting the Late Fz PCP signal. (3) Lowfat (Lft), a Ft/Ds pathway regulator, is required for the normal orientation of the Early Fz PCP signal but not the Late Fz PCP signal. (4) At the time of the Early Fz PCP signal there are symmetric gradients of dachsous (ds) expression centered on the L3 wing vein, suggesting Ds activity gradients may orient the Fz signal. (5) Localized knockdown or over-expression of Ft/Ds pathway genes shows that boundaries/gradients of Ft/Ds pathway gene expression can redirect the Early Fz PCP signal specifically. (6) Altering the timing of ds knockdown during wing development can separate the role of the Ft/Ds pathway in wing morphogenesis from its role in Early Fz PCP signaling (Hogan, 2011).

The data presented in this report allow refinement Bidirectional-Biphasic (Bid-Bip) Fz PCP signaling model, particularly the nature of the proposed Early Fz(Sple) signal (Sple is an isoform of Prickle). The Early Fz(Sple) signal is in opposing directions in the anterior and posterior wing and converges precisely at the site of the L3 vein. The site of the L3 vein, therefore, represents a discontinuity in Early Fz(Sple) signaling that is called the PCP-D (see A model for PCP specification in the Drosophila wing). However, it is clear that physical differentiation of the L3 vein is not required for the formation of the PCP discontinuity (PCP-D). The correspondence of the PCP-D with the site of the L3 vein is perhaps surprising as the compartment boundary (a barrier to clonal growth that runs a few cells anterior to the L4 vein) appears a more obvious boundary between the anterior and posterior wing. However, the L3 vein has been defined as a specific region of low Hedgehog signaling within the wing, suggesting this region has the molecular autonomy needed to function as a signaling centre. In addition, recently published work from the Eaton lab (Aigouy, 2010) has also identified the L3 vein as the boundary between oppositely polarized cells in the anterior and posterior of early pupal wings (Hogan, 2011).

Both reduced activity and uniform over-expression of Ft/Ds pathway genes have similar effects on the direction of the Fz(Sple) signal, which becomes more distal in both the anterior wing and distal regions of the posterior wing. Significantly, the Eaton lab has shown that the subcellular localization of Vang/Stbm protein in the early (15 hours a.p.f.) pupal wing of a ds mutant is more distal than wild-type in both the anterior and distal posterior wing (Aigouy, 2010). The current results are consistent with the idea that the normal direction of the Fz(Sple) signal is controlled by gradients of Ft/Ds pathway activity that can be flattened through either reduced or uniform expression of individual pathway components. An observation made by Matakatsu (2004) that ds is expressed transiently in a P-D stripe within the pupal wing blade at around the time of Early Fz PCP signaling and the peak of Ds expression has been localized to the site of the L3 vein, the same location as the wing PCP-D. This implies that there are symmetric gradients of ds expression in the anterior and posterior wing and that the Early Fz(Sple) signal points up a ds expression gradient. This conclusion is supported by the finding that the Fz(Sple) signal reorients to point away from localized ds knockdown, but not from localized ds over-expression. The Early Fz(Sple) signal also points away from over-expressed ft or fj, which suggests that Ft or Fj activity has the opposite effect to Ds activity on direction of the Fz(Sple) signal. This is the same relationship between Ft, Ds and Fj activity that has been established in the Drosophila eye. Recent molecular studies have shown that Fj, a golgi kinase, can phosphorylate cadherin domains within both Ft and Ds proteins. It has been proposed that this modification increases Ft activity, but decreases Ds activity (Hogan, 2011).

Reducing ds expression (or increasing ft or fj expression) under the control of the sal-Gal4 driver redirects the Early Fz(Sple) signal for a significant distance (ten or more cell diameters) beyond the sal-Gal4 expression domain. In principle, reducing ds expression within the sal-Gal4 domain should generate a local reversal of the ds expression gradient at the boundary of sal-Gal4 expression (e.g. the L2 vein). This short reversed ds gradient should generate a correspondingly short region of reversed Fz(Sple) signal which should be visible (on a pk^pk mutant wing) as a short region of reversed hair polarity adjacent to the L2 vein. Therefore, the propagation of reversed hair polarity significantly anterior to the L2 vein is surprising. However, a similar propagation of reversed polarity is seen adjacent to loss-of-function and over-expression clones of ds, ft or fj in the Drosophila abdomen. The model proposed for the propagation of altered polarity in the abdomen may, therefore, also apply to the Early Fz(Sple) signal in the wing (Hogan, 2011).

Since it has been established that wing hair polarity points down a gradient of Fz activity and it is proposed that the direction of the Early Fz(Sple) signal (i.e. the hair polarity that would be specified by the signal) points up a Ds expression gradient, it appears that there are opposing gradients of Ds and Fz activity during Early Fz(Sple) signaling. This relationship between Ds and Fz gradients is consistent with that described in the Drosophila eye, although it is opposite to that previously proposed in the wing. These findings, therefore, may help resolve this discrepancy between the proposed relationships of Fz and Ds activity in the eye and wing that has been highlighted by others (Hogan, 2011).

From this work it is concluded that for substantial regions of the wing (including most of the anterior wing and distal regions of the posterior wing), Ft/Ds pathway activity can be altered such that the Early Fz(Sple) signal is redirected, but the Late Fz(Pk) signal remains unaffected. For any specific experiment, this result might be explained by the specific properties of the mutant allele used or by the specific spatial or temporal activity of the Gal4 driver used to drive gene knockdown or over-expression. However, this study has shown that numerous alleles, as well as both knockdown and over-expression, of Ft/Ds pathway genes, can redirect the Fz(Sple) signal in a similar way, without affecting the Fz(Pk) signal in the same region. This suggests that across most of the wing there is a different requirement for the Ft/Ds pathway in the Early Fz(Sple) and Late Fz(Pk) signals. Moreover, it was found that loss of the Ft/Ds pathway regulator Lft affects the Early Fz(Sple) signal, but not the Late Fz(Pk) signal. This suggests that the mechanism used by the Ft/Ds pathway to direct the Early Fz(Sple) signal differs from that used to organize the Late Fz(Pk) signal (Hogan, 2011).

What, then, is the role of the Ft/Ds pathway in the Late Fz(Pk) signal? Since the Late Fz(Pk) signal organizes hair polarity, characterizing the loss of Ft/Ds pathway activity on hair polarity should be informative. It was found that driving ft or ds RNAi uniformly in the wing results in altered wing morphology, but only localized proximal hair polarity changes. This might be due to incomplete gene knockdown, coupled with different requirements for Ft/Ds activity for Late Fz PCP signaling in different regions of the wing. However, it is suggestive that wings homozygous for a fj amorphic allele show only a localized hair polarity phenotype in this same proximal region, implying that Fj is only required for hair polarity in the proximal wing. These results raise the possibility the Ft/Ds pathway is normally only required for hair polarity in the proximal wing (Hogan, 2011).

Since neither ft nor ds null flies are adult viable, previous studies have inferred the role of Ft and Ds in wing hair polarity from analyzing phenotypes of viable hypomorphic alleles, clones of amorphic alleles and localized over-expression. Some hypomorphic ds allele combinations display extensive wing hair polarity disruptions, although the residual activity of these specific alleles has not been well characterized. Wing clones homozygous for amorphic ft or ds alleles can show hair phenotypes, although this is dependent upon the position and/or size of the clone. However, mutant clones generate ectopic Ft or Ds activity boundaries/gradients in the wing and it is known that localized mis-expression of Ft/Ds pathway genes can generate hair phenotypes in wing regions not affected by uniform over-expression. Most telling, clones of fj affect hair polarity in regions of the wing that are not affected in amorphic fj wings. These results clearly show that mis-regulated Ft/Ds activity can change wing hair polarity. However, they do not definitively establish a role for Ft/Ds pathway in the normal organization of hair polarity outside of the proximal wing. Therefore, it remains possible that Ft/Ds pathway activity is only required for hair polarity in the proximal wing, but mis-regulated Ft/Ds pathway activity can induce changes in hair polarity in other wing regions. This may restrict the normal role of the Ft/Ds pathway to organizing the Late Fz(Pk) signal in the proximal wing alone (Hogan, 2011).

According to the Bid-Bip model, the two Fz PCP signaling events aligned with different axes of the developing wing allow membrane ridges to be organized in different directions in the anterior and posterior. The ability of the insect wing to deform specifically is vital for insect flight and it has been proposed that wing membrane structure helps provide the appropriate wing rigidity and flexibility. In the case of membrane ridges, the membrane should be flexible parallel to the ridges, but be resistant to folding perpendicular to the ridges. The A-P ridges in the anterior wing are perpendicular to longitudinal wing veins which suggests a rigid anterior wing structure, whereas the posterior ridges are almost parallel with longitudinal wing veins suggesting a more flexible posterior wing structure. This organization is typical for Dipteran wings which usually have a well-supported leading edge and a flexible trailing edge. Indeed, similar ridge organization have been seen in wings of other Drosophila species. Therefore, the different orientation of ridges in the anterior and posterior wing may have a functional basis. The reason for the uniform distal hair polarity across the Drosophila wing is not well understood, but is conserved in a wide range of Dipteran species suggesting a functional constraint. Therefore, the two Fz PCP signals in different directions during Drosophila wing development may provide a mechanism that allows hairs and ridges to be organized appropriately using a single signaling pathway (Hogan, 2011).

Are multiple Fz PCP signaling events active in other Drosophila tissues besides the developing wing? Intriguingly, the Prickle isoforms, Pk and Sple, play different roles in PCP in numerous Drosophila tissues, including the wing, eye, abdomen and leg. This raises the possibility that there are multiple Fz PCP signals involving differential use of Pk and Sple isoforms in each of these tissues. However, the specific phenotypes associated with loss of either or both isoforms within the different tissues suggest that the details of the Bid-Bip model are unlikely to hold true for all tissues. How can multiple Fz PCP signals occur in different directions in the same developing tissue? One possibility is that changes in the molecular makeup of the Fz PCP pathway allow it to respond to different global signals within the tissue, or to respond in different ways to the same global signal. In the Drosophila wing, this might result from the differential use of the Pk and Sple isoforms. Alternatively, the individual Fz PCP signals may respond to different global signals present at different times during tissue development or to a single dynamic global cue. The significance of Prickle isoform switching and the possibility of dynamic global PCP signals are ongoing topics of interest (Hogan, 2011).

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).

Collective polarization model for gradient sensing via Dachsous-Fat intercellular signaling

Dachsous-Fat signaling via the Hippo pathway influences proliferation during Drosophila development, and some of its mammalian homologs are tumor suppressors, highlighting its role as a universal growth regulator. The Fat/Hippo pathway responds to morphogen gradients and influences the in-plane polarization of cells and orientation of divisions, linking growth with tissue patterning. Remarkably, the Fat pathway transduces a growth signal through the polarization of transmembrane complexes that responds to both morphogen level and gradient. Dissection of these complex phenotypes requires a quantitative model that provides a systematic characterization of the pathway. In the absence of detailed knowledge of molecular interactions, this study took a phenomenological approach that considers a broad class of simple models, which are sufficiently constrained by observations to enable insight into possible mechanisms. Two modes are proposed of local/cooperative interactions among Fat-Dachsous complexes, which are necessary for the collective polarization of tissues and enhanced sensitivity to weak gradients. Collective polarization convolves level and gradient of input signals, reproducing known phenotypes while generating falsifiable predictions. A construction of a simplified signal transduction map allows a generalization of the positional value model and emphasizes the important role intercellular interactions play in growth and patterning of tissues (Mani, 2013).

Signal transduction by the Fat cytoplasmic domain

The Drosophila protocadherin Fat (Ft) regulates growth, planar cell polarity (PCP) and proximodistal patterning. A key downstream component of Ft signaling is the atypical myosin Dachs (D). Multiple regions of the intracellular domain of Ft have been implicated in regulating growth and PCP but how Ft regulates D is not known. Mutations in Fbxl7 (CG4221), which encodes an F-box protein, result in tissue overgrowth and abnormalities in proximodistal patterning that phenocopy deleting a specific portion of the intracellular domain (ICD) of Ft that regulates both growth and PCP. Fbxl7 binds to this same portion of the Ft ICD, co-localizes with Ft to the proximal edge of cells and regulates the levels and asymmetry of D at the apical membrane. Fbxl7 can also regulate the trafficking of proteins between the apical membrane and intracellular vesicles. Thus Fbxl7 functions in a subset of pathways downstream of Ft and links Ft to D localization (Bosch, 2014).

The protocadherin Ft lies at the apex of multiple pathways that together regulate growth, several aspects of PCP, and proximodistal patterning. The mechanism by which Ft functions as a signaling molecule remains poorly understood. This study has identified the F-box protein Fbxl7 as an immediate effector of Ft, that functions to restrict the levels of the atypical myosin D at the apical membrane as well as its distribution around the perimeter of the cell. In addition, Fbxl7 can regulate levels of Ft at the apical membrane (Bosch, 2014).

Recent studies have revealed that Ft's effects on distinct pathways may be genetically separated, and that multiple effector domains can contribute to the same function. Indeed, the growth-suppressing function of Ft may occur via at least two regions of the Ft ICD. One or more regions between amino acids 4834 and 4899 in full-length Ft appear responsible for Ft's ability to regulate Hippo signaling. Several mutations within this region compromise this function of Ft and cause massive tissue overgrowth (Bossuyt, 2013). Intriguingly, an allele of ft, ft⁶¹, which harbors such a mutation, showed neither an effect on the recruitment of Fbxl7 to the apical membrane nor on the binding of Ft to Fbxl7. Thus, signaling via this region of the ICD appears to be independent of Fbxl7. A second, more C-terminal region of the Ft ICD (Region D) that extends between amino acids 4975 and 4993 of full-length Ft, is removed by the ftΔD deletion and also has a growth-suppressive function albeit weaker than that of HM. This second growth-suppressive pathway requires the function of Fbxl7, as the protein generated by the ftΔD allele cannot bind to Fbxl7 nor can it localize Fbxl7 to the apical membrane. Additionally, the phenotypic abnormalities of null alleles of ft rescued by ftΔD are very similar, if not identical to those of Fbxl7 mutants. Furthermore, like ftΔD, Fbxl7 mutations do not display overt abnormalities of hair orientation in the wingor abdomen (Bosch, 2014).

Hyperactivation of the 'weaker' Fbxl7-dependent pathway can overcome the absence of the ‘stronger' Fbxl7-independent pathway; overexpression of Fbxl7 can suppress the overgrowth of ft⁶¹. Thus, while these two pathways can be dissociated at the level of the Ft ICD, they nevertheless seem to converge further downstream. This point of convergence likely involves Dachs (D) since the overgrowth of ft mutant tissue can be suppressed completely by eliminating D function. Indeed, it has previously been suggested that Ft regulates growth by restricting the levels of apical D, and regulates PCP by influencing the planar asymmetry of apical D (Bosch, 2014).

Another key finding in these experiments is that Fbxl7 mutations perturb the distribution of D around the perimeter of the apical region of the cell. D is normally biased towards the distal edge of the cell; in Fbxl7 mutants, D is more evenly distributed around the cell perimeter. The asymmetric localization of D depends on at least two different regions of Ft (Pan, 2013). One is the region that binds to Fbxl7 (Region D) and the other is composed of the last three amino acids at the C-terminus of the protein (Region F), which is not necessary for Fbxl7 localization to the apical membrane. Thus, for the regulation of D asymmetry as well, there appears to be an Fbxl7-independent pathway. The existence of multiple downstream effector pathways that converge on common biological outcomes suggests that these pathways might function redundantly to some extent and thus provide robustness. This might also explain why the phenotypes elicited by overexpression of Fbxl7 are, in general, more severe than those observed in loss-of-function mutations (Bosch, 2014).

Previous observations of the localization of Ft, Ds, and D to vesicles are suggestive of trafficking events being involved in Ft signaling. It was therefore demonstrated that, in addition to the apical membrane, Fbxl7 localizes to vesicles. Moreover, FLAG-Fbxl7 vesicles can contain Ft, Ds and D, and these may be related to the apical puncta observed on cell edges. This localization is likely specific, since no Fbxl7 co-localization is seen with other cell surface proteins such as Crumbs, Notch, and E-cadherin. Currently very little is known about the role of each of these proteins in vesicles. However, there is an increasing appreciation that most transmembrane proteins, and even proteins that are associated with the inner leaflet of the cell membrane are maintained at the plasma membrane by a dynamic process involving endocytosis and vesicle recycling (Bosch, 2014).

Evidence is provided that Fbxl7 regulates Ft apical localization, but how this regulation relates to the Fbxl7 phenotypes is not clear. Since Fbxl7 overexpression increases Fat signaling, and rescues the overgrowth-inducing ft⁶¹ allele, perhaps this is due to the increased levels of Ft protein at the apical membrane. However, Ft levels are slightly elevated in Fbxl7 mutants, which display mild overgrowth. Therefore the mutant phenotype cannot be explained by the effect on Ft. Another known regulator of apical Ft levels is lowfat (lft). Fbxl7 and Lft appear to regulate Ft in different ways. Lft overexpression, like Fbxl7, increases Ft levels. However, while Ft levels are decreased in lft mutant cells, Ft levels are increased in Fbxl7 mutant cells, though less so compared to Fbxl7 overexpression. Interestingly, for many proteins that regulate cellular trafficking, similar phenotypic abnormalities are observed with gain-of-function and loss-of-function mutations, since the normal execution of the process requires the protein to shuttle efficiently between two states. Thus dynamic aspects of the localization of Ft, Ds and D clearly merit more attention (Bosch, 2014).

The interactions observed between Fbxl7 and the adapter protein Cindr may provide clues for how Fbxl7 regulates D localization. Fbxl7-associated vesicles show almost complete overlap with GFP-Cindr and Fbxl7 can re-localize Cindr from the apical membrane to the interior of the cell. This finding, together with the observed increase in basal levels of D upon Fbxl7 overexpression, suggests that Fbxl7 may function to regulate D trafficking in a similar manner. Cindr and its mammalian orthologues Cin85 and CD2AP are thought to regulate interactions between membrane proteins and actin cytoskeleton. D is an atypical myosin with a predicted actin binding domain in its conserved head domain. Therefore, the vesicles which Fbxl7 associates with D and Cindr may be linked to the actin cytoskeleton. In addition, the finding of partial colocalization of Fbxl7 with retromer components further supports the possibility that Fbxl7 may have a role in protein trafficking (Bosch, 2014).

Many F-box proteins associate with Skp1 and Cul1 to form an SCF E3 ubiquitin ligase complex. Recruitment of specific substrates results in their poly-ubiquitylation and degradation, or mono-ubiquitylation, which can have non-degradative signaling roles. In addition, some F-box proteins have SCF-independent roles. Fbxl proteins are thought to recruit substrates to the SCF complex through the interaction with their LRR domains, and substrates have been identified for several Fbxls such as Skp2 (Fbxl1), which degrades p27. However many, like Fbxl7, are still uncharacterized as 'orphan' F-box proteins with no known substrates (Bosch, 2014).

Since this study found that Fbxl7 associates with Skp1 and Cul1, its potential substrates may be involved in Ft signaling. Fbxl7 has one described substrate in mice, Aurora A. However it is not believed Aurora A is a relevant substrate in Drosophila, as no Ft signaling defects are observed when Aurora A is knocked down or overexpressed. The identification of F-box protein substrates has mainly been accomplished by unbiased approaches. Similarly, a combination of unbiased approaches, involving proteomics, genetic interaction screens, and identifying proteins that co-localize with Fbxl7 in vesicles could be used to identify Fbxl7 substrates (Bosch, 2014).

The atypical cadherin Fat directly regulates mitochondrial function and metabolic state

Fat (Ft) cadherins are enormous cell adhesion molecules that function at the cell surface to regulate the tumor-suppressive Hippo signaling pathway and planar cell polarity (PCP) tissue organization. Mutations in Ft cadherins are found in a variety of tumors, and it is presumed that this is due to defects in either Hippo signaling or PCP. This study shows Drosophila Ft functions in mitochondria to directly regulate mitochondrial electron transport chain integrity and promote oxidative phosphorylation. Proteolytic cleavage releases a soluble 68 kDa fragment (Ft^mito) that is imported into mitochondria. Ft^mito binds directly to NADH dehydrogenase ubiquinone flavoprotein 2 (Ndufv2), a core component of complex I, stabilizing the holoenzyme. Loss of Ft leads to loss of complex I activity, increases in reactive oxygen species, and a switch to aerobic glycolysis. Defects in mitochondrial activity in ft mutants are independent of Hippo and PCP signaling and are reminiscent of the Warburg effect (Sing, 2014).

Vamana couples fat signaling to the Hippo pathway

The protocadherins Dachsous and Fat initiate a signaling pathway that controls growth and planar cell polarity by regulating the membrane localization of the atypical myosin Dachs. How Dachs is regulated by Fat signaling has remained unclear. This study identified the vamana gene (CG10933; FlyBase name Dachs ligand with SH3s or Dlish) as playing a crucial role in regulating membrane localization of Dachs and in linking Fat and Dachsous to Dachs regulation. Vamana, an SH3-domain-containing protein, physically associates with and co-localizes with Dachs and promotes its membrane localization. Vamana also associates with the Dachsous intracellular domain and with a region of the Fat intracellular domain that is essential for controlling Hippo signaling and levels of Dachs. Epistasis experiments, structure-function analysis, and physical interaction experiments argue that Fat negatively regulates Dachs in a Vamana-dependent process. These findings establish Vamana as a crucial component of the Dachsous-Fat pathway that transmits Fat signaling by regulating Dachs (Misra, 2016).

Coordinated growth and morphogenesis is critical to the development of tissues of specific size and shape. Dachsous (Ds)-Fat signaling (henceforth, Fat signaling) controls both growth, through regulation of Hippo signaling, and morphogenesis, through regulation of planar cell polarity (PCP). Fat signaling regulates Hippo signaling and PCP by controlling the membrane localization of the atypical myosin protein Dachs. Many studies have provided important insights into both how Dachs influences Hippo signaling, and how it influences PCP. In contrast, the mechanism by which Fat signaling actually controls Dachs has remained less well understood (Misra, 2016).

Fat and Ds are atypical cadherins with novel intracellular domains (ICD), which localize to the plasma membrane just apical to the adherens junctions. Fat and Ds bind to each other in a heterophilic manner, and this interaction is modulated by the Golgi-resident kinase, Four-jointed (Fj), which phosphorylates their extracellular domains. This heterophilic binding, together with the graded expression of Ds and Fj, contribute to polarization of Ds and Fat localization within cells. Three different ways by which Fat signaling influences Hippo signaling have been described: Fat signaling influences the membrane localization of Expanded (Ex) , the levels of Wts protein, and the interaction of Wts with its cofactor Mats. Each of these effects on Hippo signaling depends upon Dachs. Fat signaling affects PCP in at least two ways: through an influence on junctional tension, and by regulating the Spiny-legs (Sple) isoform of the prickle locus. Both of these effects also involve Dachs (Misra, 2016).

Dachs was identified as a key downstream effector of Fat signaling because mutations in dachs completely suppress the overgrowth induced by fat mutations, and partially suppress the PCP defects induced by fat mutations. Dachs localizes to the cell membrane just apical to the adherens junction in a polarized manner; in the developing wing Dachs is localized to the distal sides of the cell, in response to the proximal-distal gradients of Ds and Fj expression. Dachs membrane localization requires a palmitoyltransferase encoded by approximated (app), but how App influences Dachs localization is unknown. In fat or ds mutants increased levels of Dachs are observed at the apical membrane and Dachs is no longer polarized. Forcing Dachs membrane localization by fusing it to Zyxin phenocopies fat mutants. Conversely, overexpression of full-length Fat or even just the Fat intracellular domain (ICD) displaces Dachs from the membrane into the cytoplasm. These and other observations have indicated that Fat regulates growth by modulating the levels of Dachs at apical membranes, and regulates Dachs-dependent PCP by directing Dachs asymmetry (Misra, 2016).

To understand how Fat functions, several studies have examined the roles of different regions of the Fat ICD. These studies identified two regions that mediate its growth-suppressive function. One, the D region, around amino acids 4,975 to 4,993, makes a modest contribution to Hippo pathway regulation, as when this region is deleted flies are viable but their wings are approximately 30% larger than normal, and also rounder than normal. The D region is required for interaction with the ubiquitin ligase, Fbxl7, which reduces Dachs membrane levels, and mutation of which results in phenotypes similar to deletion of the D region. A second region, which has been referred to as HM, Hpo, or H2, is defined by observations that deletions within this region block the ability of Fat to activate Hippo signaling. Two alleles of fat, fat⁶¹ and fat^sum, have also been identified that harbor mutations within this region, and are associated with tissue overgrowth comparable with that caused by fat null mutations. However, the mechanism by which this region, which for simplicity is referred to as the H region, regulates the Hippo pathway, and whether it affects Dachs, are unknown (Misra, 2016).

This study reports the isolation and characterization of the Src homology 3 (SH3)-domain-containing protein encoded by vamana (vam). Loss of vam function decreases growth, whereas overexpression of vam promotes growth. These effects are mediated through regulation of the Hippo pathway, and vam functions genetically downstream of fat, as vam mutations can suppress both growth and PCP phenotypes of fat. Vam localizes to the apical region of epithelial cells in a polarized manner, co-localizing with Dachs, and is required for normal membrane localization of Dachs. Vam physically associates with the carboxy-terminal domain of Dachs and the ICDs of Ds and Fat, and is regulated by the H region of the Fat ICD. These observations identify Vam as a key mediator of signaling from Fat to Dachs (Misra, 2016).

These studies identified the C-terminal region of Dachs as sufficient to mediate its interaction with Vam. Interestingly, the original dachs allele, described almost a century ago by Bridges and Morgan (1919), is a hypomorphic allele associated with insertion of a blood transposon just upstream of the C-terminal region. Hence this allele likely encodes a truncated protein that lacks the Vam-interaction domain. Consistent with this inference, the vam null phenotype appears similar to the dachs¹ phenotype. Thus, a requirement for interaction with Vam can explain the basis for the original identification of dachs (Misra, 2016).

Vam is evolutionarily conserved among insects but with no close homologs in vertebrates. This is consistent with the fact that Dachs is also only found in insects, and the sequence of the H region is not conserved in vertebrate Fat genes. Nonetheless, Vam is structurally related to a broad family of SH2- and SH3-domain-containing proteins exemplified by CRK, Grb2, Myd88, and NCK. These proteins are referred to as signal-transducing adapter proteins and facilitate formation of protein complexes that play key roles in signal transduction. Vam is composed of just three SH3 domains; this domain organization is most similar to that of the NCK family of adapters, which contain three SH3 domains along with one SH2 domain. The finding that Vam uses both SH3-1 and SH3-3 to interact with Fat and Ds is also reminiscent of NCK family adapters, as they engage effectors using multiple SH3 domains. The Drosophila ortholog of NCK, dreadlocks (dock), interacts with cell-adhesion molecules encoded by hibris, kirre, roughest, and sticks and stones (sns) to regulate actin polymerization and growth cone migration, and functional redundancy of SH3 domains has been observed for dock. Multiple SH3 domains are also commonly observed in proteins involved in vesicular trafficking. The observation that in vam mutants Dachs accumulates in cytoplasmic puncta that could be vesicular structures suggests that Vam might influence the trafficking of Dachs (Misra, 2016).

Fat and Ds proteins are conserved in vertebrates, where they play important roles in controlling PCP, and have also been proposed to influence Hippo signaling. In the absence of a Dachs homolog, however, it has been unclear how downstream signaling is mediated in vertebrate Ds-Fat pathways. The discovery that Vam links Ds and Fat to downstream signaling raises the possibility that a different member of the signal-transducing adapter proteins could mediate downstream Ds-Fat signaling in vertebrates (Misra, 2016).

The H region of the Fat ICD plays a crucial role in Hippo pathway regulation. This analysis of Fat ICD truncations revealed that the H region inhibits Vam and Dachs membrane accumulation, the influence of Fat ICD deletions on Hippo signaling correlates with their influence on Vam and Dachs membrane localization, and the H region of Fat can associate with Vam. Together with observations that Vam associates with and regulates Dachs, these observations lead to the inferrence that the H region normally functions to promote Hippo signaling through its association with, and regulation of, Vam. Fat also influences growth and Dachs accumulation through a second region of the ICD, the D region, which interacts with Fbxl7. Because mutation of the D region, or mutations in Fbxl7, have weaker phenotypes than mutations in the H region, the H region appears to play the larger role in Dachs regulation, but nonetheless it is expected that both regions normally act in parallel to regulate membrane levels of Dachs and thus, ultimately, Hippo signaling (Misra, 2016).

The localization of Vam in different genotypes, together with its physical interactions, suggests models for how Vam regulates Dachs localization. Since Vam and Dachs are reciprocally required for each other's membrane localization, it is inferred that a complex between these two proteins is required for their stable localization to apical junctions, where Dachs regulates PCP (via interactions with Sple) and Hippo signaling (via interactions with Zyxin and Warts). The observations that Fat promotes removal of Vam and Dachs from the subapical membrane, associates with Vam, yet does not visibly co-localize with Vam at apical junctions, suggests that Fat normally removes Vam-Dachs complexes from the subapical membrane. One mechanism by which this might occur is through binding of Fat to Vam, followed by endocytosis of Fat-Vam-Dachs complexes. Alternatively, Fat binding might disrupt Vam-Dachs binding, as these proteins normally do not localize to the membrane in isolation (Misra, 2016).

It was also observed that Vam can interact with the Ds ICD, and that it does so through the same SH3 domains as it uses to interact with the Fat ICD. This suggests that these interactions are likely to be competitive. In this case, interaction of Vam with the Ds ICD could promote Vam and Dachs membrane localization by opposing the influence of Fat on Vam. For example, by competing with Fat for binding to Vam, Ds could prevent Fat from disrupting Vam-Dachs interactions, or promoting endocytosis of a Vam-Dachs complex. Consistent with this suggestion that the Ds ICD stabilizes Vam and Dachs at apical junctions, Vam, Ds, and Dachs normally all co-localize in puncta on the distal side of wing cells. The ability of Vam to associate with the ICDs of both Fat and Ds could thus provide a simple mechanism explaining how the ICD of Ds seems to promote Dachs membrane localization, whereas the ICD of Fat inhibits it (Misra, 2016).

The novel SH3 domain protein Dlish/CG10933 mediates fat signaling in Drosophila by binding and regulating Dach

Much of the Hippo and planar cell polarity (PCP) signaling mediated by the Drosophila protocadherin Fat depends on its ability to change the subcellular localization, levels and activity of the unconventional myosin Dachs. To better understand this process, a structure-function analysis of Dachs was performed, and this was used to identify a novel and important mediator of Fat and Dachs activities, a Dachs-binding SH3 protein that has been named Dlish (Dachs ligand with SH3s). Dlish was found to be regulated by Fat and Dachs. Dlish also binds Fat and the Dachs regulator Approximated, and Dlish is required for Dachs localization, levels and activity in both wild type and fat mutant tissue. The evidence supports dual roles for Dlish. Dlish tethers Dachs to the subapical cell cortex, an effect partly mediated by the palmitoyltransferase Approximated under the control of Fat. Conversely, Dlish promotes the Fat-mediated degradation of Dachs (Zhang, 2016).

Heterophilic binding between the giant Drosophila protocadherins Fat and Dachsous (Ds) both limits organ growth, via regulation of the Hippo pathway, and orients planar cell polarity (PCP), through cell-by-cell polarization of Fat, Ds and their downstream effectors. Loss of Fat and, to a lesser extent, Ds, leads to the profound overgrowth of the Drosophila imaginal discs that give rise to adult appendages, and loss of either disorders the polarity of cell divisions, hairs and other morphological features in a variety of Drosophila tissues. But while players and pathways have been defined that are genetically downstream of Fat-Ds binding, only a little is known about the biochemical links between these and their most powerful regulator, the intracellular domain (ICD) of Fat (Zhang, 2016).

A good deal of the recent work on Fat effectors has focused on the regulation of unconventional type XX myosin Dachs. Dachs is critical first because it provides the only known marker specifically sensitive to changes in the Fat/Ds branches of both the Hippo and PCP pathways. Dachs is normally concentrated in the subapical cell cortex, overlapping subapically-concentrated Fat and Ds. Loss of Fat greatly increases subapical Dachs levels, and polarization of Fat and Ds to opposite cell faces can in turn polarize Dachs to the face with less Fat. Fat thus inhibits or destabilizes subapical Dachs, while Ds may do the opposite. Downstream changes in Hippo or PCP activities do not affect Dachs (Zhang, 2016).

Dachs changes are also critical because they play a major role downstream of Fat. Dachs binds to and inhibits the activity of the kinase Warts (the Drosophila Lats1/2 ortholog), both reducing Warts levels and changing its conformation. Warts is concentrated in the subapical cell cortex, and thus the increased cortical Dachs of fat mutants should reduce the phosphorylation of Yorkie by Warts, allowing Yorkie to move into the nucleus to drive the transcription of growth-promoting target genes. Indeed, Dachs is necessary for the overgrowth and increased Yorkie target gene expression of fat mutants. Dachs overexpression also causes overgrowth, although more weakly than the overgrowth caused by the loss of Fat, indicating that Dachs is partly sufficient (Zhang, 2016).

Dachs can also bind to the core PCP pathway component Spiny legs (Sple) and alter its localization, thus influencing PCP in the subset of tissues that rely on Sple. The increased levels of unpolarized Dachs in fat mutants may misdirect Sple, accounting for at least some of the PCP defects; fat mutant hair PCP defects are improved, although not eliminated, by loss of dachs (Zhang, 2016).

Dachs has not been shown to interact directly with Fat's ICD, and only three other proteins are known to affect Dachs accumulation in the subapical cell cortex, the casein kinase ε Discs overgrown (Dco), Approximated (App) and F-box-like 7 (Fbxl7). Dco may act through Fat itself: Dco binds and phosphorylates the Fat ICD, and loss of Dco function causes strong overgrowth and increases subapical Dachs, similar to loss of Fat (Zhang, 2016).

App suggests a mechanism in which Fat inhibits the tethering of Dachs to protein complexes in the subapical domain. app mutants decrease subapical Dachs levels and reduce Dachs activity. Thus, like dachs mutants, app mutants reverse the overgrowth and increased Yorkie target gene expression normally observed in fat mutants, and improve hair PCP. App is one of 20 Drosophila DHHC palmitoyltransferases, transmembrane proteins responsible for adding palmitates to cytoplasmic proteins and thereby anchoring them to cell membranes. App is also concentrated in the subapical cell membrane and can bind both Dachs and the Fat ICD. Thus, in the simplest model App palmitoylates or tethers Dachs, concentrating it in the cell cortex, and Fat works in part by sequestering or inhibiting App. However, Dachs is not detectably palmitoylated (Zhang, 2016).

This study describes the function of a novel Dachs binding protein, and shows that its effects provide strong evidence for both the palmitoylation-dependent and degradation-dependent regulation of Dachs. Structure-function analysis of Dachs found regions required for its normal subapical localization, and this information was used as the basis for a screen for novel Dachs binding partners. A direct binding partner was found for the Dachs C-terminus, the previously uncharacterized SH3 domain protein CG10933, which has been renamed Dachs ligand with SH3s, or Dlish. The activity and subapical concentration of Dlish are regulated by Fat, Dco and Dachs, and Dlish in turn is required for the subapical concentration and full activity of Dachs in both wild type and fat mutant cells. Dlish localization also depends on App; furthermore Dlish binds to and is palmitoylated by App, and palmitoylation can be suppressed by Fat. Loss of Dlish also increases the total levels of Dachs, likely by blocking Fat-mediated destabilization of Dachs. It is proposed that Dlish targets Dachs to subapical protein complexes in part via Fat-regulated, App-mediated palmitoylation. Dlish thereby concentrates Dachs where it can efficiently inhibit subapical Warts, and conversely links Dachs to the machinery for Fat-dependent destabilization (Zhang, 2016).

The unconventional myosin Dachs is an important effector Fat/Ds-regulated Hippo signaling, as its heightened subapical levels in fat mutants inhibit and destabilize Warts, freeing Yorkie to increase the expression of growth-promoting genes. A structure-function analysis of Dachs was used as a springboard to search for new binding partners that are critical for Dachs localization and function, and have found Dlish (CG10933), a novel SH3 domain protein. Dlish binds directly to the Dachs C-terminus; loss of Dlish disrupts Dachs localization, levels and function: subapical accumulation of Dachs is reduced and cytoplasmic and total levels increase, both in wild type and fat mutant tissue, while activity is lost. Importantly, Dlish is regulated by Fat, as loss of Fat greatly increases Dlish levels in the subapical cell cortex and, like Dachs, Dlish is needed for much of the fat mutant overgrowth (Zhang, 2016).

Dlish also binds the ICD of Fat and other Fat-binding proteins, including two that likely mediate part of its function: the palmitoyltransferase App and the F-box protein Fbxl7. Thus Dlish provides a new biochemical link from the Fat ICD to Dachs regulation. Evidence indicates that Dlish plays two different and opposing roles (see Models of Fat-mediated regulation of subapical Dachs by Fat-inhibited subapical tethering and Fat-stimulated destabilization). First, it helps tether Dachs in the subapical cell membrane, in part via Fat-regulated, App-dependent palmitoylation, so that Dachs can more efficiently inhibit Warts. Second, it links Dachs to Fat-organized machinery for Dachs destabilization, including Fbxl7, and thus helps reduce Dachs levels (Zhang, 2016).

Dlish and Dachs cooperate to target or tether the Dlish-Dachs complex, as each is necessary, and to a weaker extent sufficient, for the subapical concentration of the other. The Dachs contribution is likely through tethering the complex to the cortical cytoskeleton, as this study found that loss of the F-Actin-binding myosin head blocks the subapical localization of Dachs. This would agree with recent biochemical analyses that suggest that Dachs has no motor function, acting rather as an F-Actin-binding scaffolding protein (Zhang, 2016).

The Dlish contribution, on the other hand, depends at least in part on its ability to bind the transmembrane DHHC palmitoyltransferase App. Loss of App and Dlish have very similar effects on Dachs localization and activity. This study found that loss of App disrupts the subapical accumulation of Dlish in vivo and that App can stimulate palmitoylation of Dlish in vitro. Thus, palmitoylation of Dlish likely stimulates membrane association of both Dlish and its binding partner Dachs (Zhang, 2016).

App also has additional effects on Fat pathway activity. First, App has palmitoyltransferase-independent activity and can co-IP Dachs in vitro. Thus, while palmitoylation of Dlish may mediate some of App's activity, subapical App may simultaneously help localize the Dlish-Dachs complex by physical tethering. And while both palmitoylation and tethering of the Dlish-Dachs complex is likely critical for the fat mutant phenotype, App also has a function that depends on the presence of Fat, as App can bind, palmitoylate and inhibit the activity of Fat's ICD (Zhang, 2016).

An important question is whether the absence of Fat regulates the App-dependent tethering of the Dlish-Dachs complex. The Fat ICD can complex with both Dlish and App. Dlish and App can bind not only the C-terminal region of the Fat ICD where Fat is palmitoylated, but also the PH and Hippo domains which was shown to played the strongest role in Dachs regulation. An attractive mechanism is that Fat inhibits the interaction between App and Dlish, reducing App's ability to palmitoylate and tether the Dlish-Dachs complex. In the absence of Fat, App and Dlish are freed to tether Dachs, and Dachs now inhibits and destabilizes Warts, causing overgrowth. In support of this model, it was found that overexpression of Fat's ICD in vitro can reduce App-stimulated palmitoylation of Dlish (Zhang, 2016).

The evidence further indicates that Dlish targets Dachs for Fat-dependent destabilization. Loss of Fat increases not only subapical Dachs, but also total Dachs levels. In the presence of Dlish the increased Dachs remains subapical. Loss of Dlish also increases the total levels of Dachs, but now that increase is cytoplasmic, and much less effective at inhibiting Warts. These Dachs increases are unlikely to have independent causes, as they are not additive; total Dachs levels are similar after loss of Fat, Dlish or both (Zhang, 2016).

It is proposed that in wild type cells there is a flux of the Dachs-Dlish complex from the cytoplasm to the subapical cell cortex, where a Fat-dependent complex destabilizes Dachs. Normally the tethering effects of Dlish predominate over the Fat-dependent destabilization, and moderate levels of subapical Dachs are maintained. Destabilization is lost without Fat; this combines with Dlish-mediated tethering to increase subapical Dachs. Without Dlish the subapical tethering of Dachs is disrupted, access to Fat-dependent destabilization is lost and the now cytoplasmic Dachs increases. The model thus explains why the excess, largely cytoplasmic Dachs caused by reduced Dlish function is not greatly influenced by the presence or absence of Fat (Zhang, 2016).

In addition to any effects caused by changing the subcellular localization of Dachs, Dlish may also provide a direct link to the machinery for protein ubiquitination, as Dlish can co-IP with the E3 ubiquitin ligase Fbxl7, as well as the related F-box ubiquitin ligase Slimb. Fbxl7 is particularly intriguing, as it binds to and is regulated by Fat's ICD, and reduces subapical Dachs, perhaps via ubiquitination. Slimb can bind and ubiquitinate Expanded, a subapical regulator of Hippo signaling with links to Fat and Dachs function. But while it was found that loss of Fbxl7 or Slimb increases subapical Dachs and Dlish, these effects are weak, and the large increase in total Dachs levels caused by loss of Dlish or Fat must involve additional partners (Zhang, 2016).

Mutations in Fat’s closest mammalian homolog Fat4 (FatJ) and its Ds-like ligands strongly disrupt PCP-like processes, and have in humans been associated with the multisystem defects of Hennekam and Van Maldergem syndromes. There has been some debate, however, about whether the mammalian proteins retain direct regulation of Hippo activity. Nonetheless, Fat4 has been linked to Hippo changes in both normal development and tumors, mutations in Fat4 or Dachsous1 change the balance of precursors and mature neurons in the developing neuroepithelium of both humans and mice, and the mouse defect can be reversed by knockdown of the Yki homolog Yap. But the mechanisms underlying these effects are unknown, and Fat4 cannot regulate Hippo signaling in Drosophila (Zhang, 2016).

It is therefore important to note that while homologs of Dachs and Dlish are found throughout the animal kingdom, they are apparently absent from vertebrates. This suggests that the Dachs-Dlish branch of the Fat-Ds pathway, with its powerful effect on Warts activity, is also lacking. Nonetheless, it has been suggested that Drosophila Fat and Ds can affect Hippo pathway activity in a Dachs-independent manner. It is also clear that Drosophila Fat has Dachs-independent effects on PCP; indeed the N-terminal 'PCP' domain of the Fat ICD that did not affect Dachs in this study is sufficient to improve the PCP defects of fat mutants. These or alternative pathways may still be present in mammals (Zhang, 2016).

DEVELOPMENTAL BIOLOGY

In discs of mature larvae the expression of the ft gene, detected by in situ hybridization with a ft specific probe, shows a spatially heterogeneous distribution. In wild-type mature wing discs, ft expression is ubiquitous but preferentially accumulates in the axillar region and in the basal posterior region, with graded differences away from these maxima. This pattern of expression is more diffuse in young imaginal discs (72-h AEL). Regions of high expression appear in the notum and in halteres in homologous regions to the wing disc. Leg and eye-antenna discs also show heterogeneous patterns of expression. Interestingly, these patterns are similar to those shown for dachsous (ds) which also encodes for a member of the cadherin superfamily of proteins (Garoia, 2000).

In ft¹⁸ (and ft⁴) overgrown discs the expression of ft mRNA is weak and appears restricted to the distal wing pouch. Since these alleles possibly do not correspond to transcriptional nulls, the reduction of transcription in ft mutants may reflect feed back control of ft gene expression (Garoia, 2000).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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 d^GC2 mutation deletes part of the N terminal extension. As d^GC2 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. d^GC13 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, wts^P2, 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).

The tumor suppressor genes dachsous and fat modulate different signalling pathways by regulating dally and dally-like

The activity of different signaling pathways must be precisely regulated during development to define the final size and pattern of an organ. The Drosophila tumor suppressor genes dachsous (ds) and fat (ft) modulate organ size and pattern formation during imaginal disc development. Recent studies have proposed that Fat acts through the conserved Hippo signaling pathway to repress the expression of cycE, bantam, and diap-1. However, the combined ectopic expression of all of these target genes does not account for the hyperplasic phenotypes and patterning defects displayed by Hippo pathway mutants. This study identified the glypicans dally and dally-like as two target genes for both ft and ds acting via the Hippo pathway. Dally and Dally-like modulate organ growth and patterning by regulating the diffusion and efficiency of signaling of several morphogens such as Decapentaplegic, Hedgehog, and Wingless. These findings therefore provide significant insights into the mechanisms by which mutations in the Hippo pathway genes can simultaneously alter the activity of several signaling pathways, compromising the control of growth and pattern formation (Baena-Lopez, 2008).

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 Tkv^Q-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).

The DHHC palmitoyltransferase approximated regulates Fat signaling and Dachs localization and activity

Signaling via the large protocadherin Fat (Ft), regulated in part by its binding partner Dachsous (Ds) and the Golgi-resident kinase Four-jointed (Fj), is required for a variety of developmental functions in Drosophila. Ft and, to a lesser extent, Ds suppress overgrowth of the imaginal discs from which appendages develop and regulate the Hippo pathway. Ft, Ds, and Fj are also required for normal planar cell polarity (PCP) in the wing, abdomen, and eye and for the normal patterning of appendages, including the spacing of crossveins in the wing and the segmentation of the leg tarsus. Ft signaling has been shown to be negatively regulated by the atypical myosin Dachs. This study identifies an additional negative regulator of Ft signaling in growth control, PCP, and appendage patterning, the Approximated (App) protein. App encodes a member of the DHHC family, responsible for the palmitoylation of selected cytoplasmic proteins. Evidence is provided that App acts by controlling the normal subcellular localization and activity of Dachs (Matakatsu, 2008).

Crossvein spacing and tarsal leg segmentation are extremely sensitive to changes in Fat (Ft) activity; they are disrupted in weak Ft-pathway mutants that have no obvious growth or planar cell polarity (PCP) defects. Similar disruption occurs in app¹ homozygotes: The distance between the anterior crossvein (ACV) and posterior crossvein (PCV) is reduced, and one or more tarsal leg joints are lost or reduced. app¹ hemizygote wings also have weak PCP defects. Ethyl methanesulfonate was used to generate additional mutations that failed to complement app¹. Of these, app^e6 was semilethal in homozygotes and hemizygotes, and escaper adults had more extensive wing PCP defects, both proximally and in a distal region between the third and fourth longitudinal veins. They also had abdominal PCP defects: In the anterior compartment, most hairs point in the normal posterior direction, but polarity was disturbed around the anterior-posterior (A/P) boundary and extensively reversed in the posterior compartment. app^e6 appears amorphic, given that the defects were not noticeably stronger in hemizygotes (Matakatsu, 2008).

The development of PCP in the pupal wing is accompanied by the polarized redistribution of the 'core' planar-polarity proteins to the proximal, distal, or proximal and distal faces of single wing cells. PCP mutants can be separated according to their effects on this polarization. Mutations in the core PCP proteins reduce the levels and block the polarization of the other core PCP proteins, whereas changes in ft, ds, or fj expression can reorient core PCP protein polarization along inappropriate axes. The levels of the core PCP protein Flamingo (Fmi, also known as Starry night) were not reduced in app^e6 mutant clones (more than 30 examined), and Fmi polarization was in some cases reoriented. This further supports App's involvement in Ft signaling (Matakatsu, 2008).

app was mapped to a portion of 69A2-A4 containing seven known or predicted genes. app¹, app^e1, app^e3, and app^e6 all contained mutations in the 5' coding exons of one of these, the CG5620 Flybase gene prediction. A UAS-driven RNAi transgene was constructed corresponding to the 5' end of CG5620; it was expressed in developing dorsal wings by using ap-gal4; this produced app-like wing and PCP defects on the dorsal surface (Matakatsu, 2008).

However, the 3' end of the CG5620 coding prediction is in error. Products obtained by using 3' RACE contained instead the 3' exon of the adjacent CG17144 prediction, as did a BDGP EST. This transcript was called app-RA, and it was shown that the corresponding App-PA protein is produced in flies. Another EST predicts a shorter transcript called app-RB. No transcripts were found covering the entire final coding exon of CG5620 in embryonic or larval cDNA libraries or by RACE. However, the full CG5620 prediction is conserved in Drosophila pseudoobscura, suggesting that it might be utilized; this was termed app-RC (Matakatsu, 2008).

The wing and leg defects of app homozygotes were rescued by expressing UAS-app-RA, UAS-app-RB, or UAS-app-RC with either act-gal4 or en-gal4. Overexpression of higher levels of UAS-app with strong drivers such as ap-gal4 or tub-gal4 also disrupted PCP in the proximal wing and abdomen (Matakatsu, 2008).

app encodes a member of the DHHC protein family responsible for adding palmitates to cytoplasmic proteins. Eukaryotes contain multiple members of the DHHC family, with 8 predicted in yeast, 23 in mammals, and 20 in Drosophila. The region common to all predicted App isoforms contains four predicted transmembrane domains, with a DHHC cysteine-rich domain (DHHC-CRD) located between the second and third transmembrane domains. It is likely that the DHHC-CRD is located on the cytoplasmic side of the membrane, as in the yeast DHHC protein AKR1 (Matakatsu, 2008 and references therein).

Alignments using the region common to the App isoforms indicate that App is in the same subfamily as the human ZDHHCs 9, 14, and 18 and is the Drosophila protein most similar to yeast ERF2. The similarity between App and these proteins drops off at the C-terminal end of the common region. The divergent C termini of App-PA and App-PC have no significant similarity to each other or to other proteins in the database outside the drosophilids, except for a short region at the C terminus of App-PA that is similar to predicted App homologs from the insects Tribolium and Apis. App-PB has a much shorter C terminus. Because any of the putative app transcripts rescued the app leg and wing phenotypes, the different C termini are dispensable for these phenotypes (Matakatsu, 2008).

app¹ contains a missense mutation N-terminal to the DHHC-CRD domain and an aberration that introduces a frameshift predicted to truncate the C-terminal end of App-PA. app^e3 contains a missense mutation that changes a conserved cysteine in the DHHC-CRD. Because the DHHC-CRD is required for palmitoyltransferase activity, this supports a role for palmitoylation in Ft signaling. app^e6 and app^e1 contain nonsense mutations predicted to truncate the protein prior to or toward the end of the DHHC-CRD, respectively, and therefore app^e6 is likely null for App function (Matakatsu, 2008).

Two antisera were generated, one specific for App-PA and one for the common region. Both antisera uniformly stained embryos, imaginal discs, and pupal wings, and staining was lost from mitotic homozygous app^e6 clones, confirming the expression of the App-PA isoform; similar results with anti-App-common. There is no obvious asymmetric distribution of the App protein along the proximodistal or anterior-posterior axes of imaginal discs or pupal wings. However, staining was especially strong in the apical cell cortex, and this concentration did not extend more basally to the adherens junction marker DE-cadherin. This is similar to the distribution of Ft and Ds, and there is overlap between the regions where App, Ds, and Ft are concentrated. This result is surprising because human ZDHHC9, 14, and 18 and yeast ERF2 are concentrated in the Golgi or ER; only a few, less similar ZDHHCs have been detected at the plasma membrane. No significant overlap was observed between App and Golgi or ER markers in wing discs. Although App must traffic through the ER and Golgi, these results suggest that App is active in the plasma membrane, in or near the apical region where Ft and Ds are concentrated (Matakatsu, 2008).

However, App does not detectably regulate Ft and Ds levels or their binding. The binding between Ft and Ds stabilizes both proteins at the cell surface in wing discs, but app^e6 clones did not affect Ft or Ds levels or distribution. Creating artificial boundaries of ft or ds expression also strongly polarizes wing PCP, as do boundaries of the Fj kinase that phosphorylates Ft and Ds and modulates their levels. If App affected Ft-Ds levels or binding, App boundaries would be expected to affect PCP. However, small to moderately sized app^e6 clones did not affect PCP, whereas large clones only affected PCP in the regions of the wing where defects were observed in app^e6 homozygotes. There was no tendency to reorient hairs at clone boundaries, and only rarely did regions with altered PCP affect PCP in adjacent wild-type cells; these findings may be due to altered cell interactions mediated by the core polarity proteins. Sharp boundaries of UAS-app-RA misexpression (e.g., driven with the posterior driver en-gal4) also had no effect on PCP. Anti-App staining was not altered in wing discs by ft or ds clones. Thus, despite their colocalization, there is no evidence that App physically interacts for Ft or Ds (Matakatsu, 2008).

PCP defects, reduced crossvein spacing, and lost tarsal leg joints can be caused by either gains or losses in Ft signaling, so the phenotypes of app ft double mutants were examined. ft^fd and ft^G-rv are likely null alleles predicted to truncate Ft N-terminal to its transmembrane region. ft^fd homozygotes and ft^fd/ft^G-rv die during late pupal stages with strongly overgrown imaginal discs and disc-derived tissues; late pupal abdomens are not overgrown but have strong PCP defects. These phenotypes were partially rescued in ft^fd; app^e6 and ft^fd/ft^G-rv; app^e6 flies: Overgrowth and extra folding of imaginal discs were suppressed and lethality and abdominal PCP defects were reduced. PCP was still defective in proximal portions of the wing but was normal in the distal wing, in contrast to the distal defects in viable ft¹⁸ wings. Thus, App acts genetically downstream of and in opposition to Ft in both growth control and PCP (Matakatsu, 2008).

The effects of app mutations on Ft signaling are strikingly similar to those caused by reducing the function of the atypical myosin Dachs). Like app mutations, dachs (d) mutations partially suppress the overgrowth and PCP defects caused by loss of Ft. The adult phenotypes are also similar, although the defects in d null mutants are stronger than those of app^e6. Null d^GC13 hemizygotes and hypomorphic d¹ homozygotes reduce tarsal leg segmentation and the distance between the ACV and PCV and cause mild wing PCP defects that are quite similar to those observed in null app mutants. Like app^e6 clones, d^GC13 clones had PCP defects when in regions of the wing where defects occur in d homozygotes, and Fmi polarization was reoriented in d¹ pupal wings. d mutants also had abdominal PCP defects similar to those in app mutants: Polarity was almost normal in the anterior compartment, but abnormal near the A/P compartment boundary and reversed in the posterior compartment (Matakatsu, 2008).

Therefore the effects of App on the levels and distribution of a V5-tagged Dachs protein were examined. Dachs:V5 normally accumulates at higher levels in the apical cell cortex in wing imaginal discs, overlapping the region of high anti-App staining. Apical Dachs accumulation was greatly reduced, although not completely eliminated, in app^e6 clones. No changes were detected in the levels of basolateral or cytoplasmic Dachs:V5. Although App affects Dachs accumulation at the apical cell cortex, anti-App staining was normal in d mutant clones (Matakatsu, 2008).

Co-overexpression of App and Dachs:V5 greatly increased the accumulation of Dachs at the cell cortex compared with the expression of Dachs:V5 alone. Coexpression of App also increased the efficacy of Dachs in growth and PCP. Even though overexpression of App-RA did not obviously increase growth, coexpression of App-RA and Dachs caused greater overgrowth than did the expression of Dachs alone. Co-overexpression of Dachs and App caused more extreme PCP defects in the wing and abdomen than did the overexpression of App alone, whereas overexpression of Dachs:V5 alone did not affect PCP (Matakatsu, 2008).

It is likely that much or all of the app mutant phenotype is mediated by the reduction of effective Dachs at the apical cell cortex. The effects of app and d mutants are not additive: Double mutants for null app and d alleles resembled the stronger d null phenotype, as expected if App works by controlling Dachs activity. Because App affects Dachs post-transcriptionally, it is unlikely that overexpressed Dachs would fully rescue the app null. Nonetheless, overexpression of UAS-d with ap-gal4 or en-gal4 rescued the wing PCP defects normally found in the distal wing of app^e6 mutants and partly rescued the crossvein spacing and leg-joint defects of app^e6. That Dachs retains some activity in the absence of App is consistent with the low but significant levels of Dachs that remain at the apical cell cortex in app^e6 clones. Different DHHC proteins can palmitoylate the same target, so other Drosophila DHHC proteins may be supplying residual activity in the absence of App (Matakatsu, 2008).

It is concluded that App palmitoyltransferase acts in opposition to the Ft pathway, largely or wholly by controlling the apical-cell-cortex localization and the activity of the atypical myosin Dachs. This localization is probably required for full Dachs activity. For growth control, this localization would place Dachs near not only Ft, but also the Hippo pathway member Warts. Warts is concentrated near the cell cortex, with an apical bias that overlaps the region of strong App and Dachs accumulation. Dachs binds Warts and may thereby regulate the Hippo pathway, accounting for its effects on Ft-dependent growth control (Matakatsu, 2008).

Dachs also modulates the effects of Ft signaling on PCP. It is not clear whether this modulation is also mediated through changes in Warts activity. Warts is thought to act by changing the activity of the transcription factor Yorkie, an effect that would not directly confer polarity. Moreover, Dachs was reported to accumulate preferentially on the distal faces of some wing-disc cells, suggesting that Dachs is involved in cell polarization independent on any effect on transcription, probably via as-yet-unknown binding partners (Matakatsu, 2008).

It remains possible that Dachs is palmitoylated by App. However, there is no precedent for palmitoylation of a myosin, nor does Dachs score highly when an algorithm is used that detects palmitoylation sites. Nor has Dachs palmitoylation been detected by using the acylbiotin-exchange technique. The effect of App may thus be less direct, palmitoylating a binding partner or regulator of Dachs. Although Dachs is a myosin, portions of Dachs are unique and lack known protein-interaction motifs. Warts is the only proven binding partner for Dachs, but app^e6 clones did not affect the levels or cell-cortex localization of Myc-tagged Warts in wing discs (Matakatsu, 2008).

Given that the human and yeast DHHC proteins that App most resembles palmitoylate many targets, the adult phenotypes of app mutants are surprisingly specific to the Ft pathway. One known target of ERF2 and ZDHHC9 is Ras, whose activity relies on membrane localization through both farnesylation and palmitoylation. Intriguingly, the Ras and MAPK pathways interact with the Ft pathway in growth control. However, reducing Ras activity causes loss of wing veins, a phenotype not observed in app mutations, and does not cause the PCP and appendage-patterning defects of app and d mutants. Moreover, reducing Ras activity via expression of a dominant negative EGF receptor did not affect the levels of Dachs:V5 in wing discs. The different subcellular distributions of App, to the cell cortex, and ZDHHC9 and ERF2, to endomembranes, suggests they have different roles and targets, despite their strong similarity at the amino acid level (Matakatsu, 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).

Drosophila Dachsous and Fat polarize actin-based protrusions over a restricted domain of the embryonic denticle field

Atypical cadherins Dachsous (Ds) and Fat coordinate the establishment of planar polarity, essential for the patterning of complex tissues and organs. The precise mechanisms by which this system acts, particularly in cases where Ds and Fat act independently of the 'core' Frizzled system, are still the subject of investigation. Examining the deployment of the Ds-Fat system in different tissues of Drosophila, has provided insights into the general mechanisms by which polarity is established and propagated to coordinate outcomes across a field of cells. The Drosophila embryonic epidermis provides a simple model epithelia where the establishment of polarity can be observed from start to finish, and in the absence of proliferation, over a fixed number of cells. Using the asymmetric placement of f-actin during denticle assembly as a read-out of polarity, this study examined the requirement for Ds and Fat in establishing polarity across the denticle field. Comparing detailed phenotypic analysis with steady state protein enrichment revealed a spatially restricted requirement for the Ds-Fat system within the posterior denticle field. Ectopic Ds signaling provides evidence for a model whereby Ds acts to asymmetrically enrich Fat in a neighboring cell, in turn polarizing the cell to specify the position of the actin-based protrusions at the cell cortex (Lawlor, 2013).

Recent studies in the Drosophila wing and other tissues suggest that polarity may initiate at a localized signalling boundary. In this analysis of the denticle field, the examination of Ds and Fat accumulation and their loss-of- function phenotypes has suggested that a signaling boundary might also be involved. Indeed, creating an ectopic Ds focus supported that notion. Thus, across the denticle field it appears that Ds signaling from the anterior edge of column 5 cells generates an asymmetry in Fat enrichment with high levels along the posterior edge of neighboring cell column 4. This asymmetric deployment would need to be propagated to each column interface anterior (and posterior) to this. The distribution of Four-jointed coupled with its known influences on Ds-Fat binding could support this propagation. For example, since Fj is more highly expressed in column 3 compared with column 4 (see also Marcinkevicius and Zallen, 2013), that would increase the affinity of Fat from column 3 to bind Ds presented from the anterior of column 4 cells (Lawlor, 2013).

However, one difficulty in invoking a role for Four-jointed is that no phenotype was observed in fj mutants. An alternative explanation for propagation might be that since this cell field is rather small, if a strong enough bias existed that presented Ds from the column 5 side of the 4/5 interface, passive propagation could perhaps account for spread of that bias to other interfaces. The 4/5 interface indeed has special properties in several regards. First, across this interface there are signaling asymmetries in EGF receptor and Notch pathway activation. Second, tendon cell specification in column 5 cells nonautonomously influences the shape of denticles on column 4 cells. Finally, during column alignment of denticle field cells, the 4/5 interface aligns earlier and relies partly on a mechanism distinct from the columns anterior and posterior to it (Simone, 2010; Marcinkevicius, 2013). Thus, it is speculated that the 5 side of the 4/5 interface might present Ds in a manner that is unique from other interfaces and thereby sets polarization. The idea that initial polarization starts at a signaling boundary suggests a common theme between the embryonic epidermis and the much more expansive imaginal disk epithelia. Recent work in disks suggests that polarization needs to occur only over a few cell widths, and that, once established, this incipient polarization can be grown through morphogenesis, rather than continually developed by long range gradients. Thus, studying polarization in tissues that are small in scale, such as the embryonic epidermis, may contribute to understanding of the initial polarizing events that occur also in expansive tissues (Lawlor, 2013).

Prior analysis in the later larval epidermis showed that Ds and Fat acted to polarize a restricted domain of the denticle field. This idea was nicely extended by the observation that the precursor to denticles, the f-actin based protrusions (ABPs), were misplaced in the embryonic epidermis in ds and fat mutants. In this study, using quantitative analysis of ABP placement, the spatial requirement for Ds-Fat could further be characterized. Scatter plot analysis, which records the relative position of each individual ABP position, revealed that there exists a graded retention of polarity in the mutants. For instance, ds mutants exhibit a severe loss of polarity in column 5 with ABP placement appearing more and more correctly polarized as one moves anterior toward column 2. This strongly supports the idea that a second polarizing input remains in place in ds or fat mutants. That input is likely to be the Fz system, which has been shown to affect the anterior region of the denticle field. In fact, again using quantatative analysis, it was showm previously that removing fz in ds mutants leads to more severe mis-polarization of larval denticle columns 2 and 3 (Donoughe, 2011). Thus, the denticle field is polarized using input from each pathway, with the Fz system largely responsible over the anterior domain, and the Ds-Fat system responsible over the posterior. A corollary of this is that the Fz and Ds pathways provide separate inputs to planar polarity (Lawlor, 2013).

In considering how ABP assembly might be polarized several pieces of data enter into consideration. First, the ABPs did not simply exhibit binary states of 'membrane polarized', or not. Rather, the graded retention of polarization observed in mutants suggests that ABPs can be stably formed at different coordinates along the apical face of the cell. Furthermore, in cases where two or more ABPs are made by a single cell, they almost always both exhibit a similar polarity value. Thus, it will be important to understand what constitutes the cortical apical structures that capture the ABPs. Those structures must interact with the effector circuit for Ds-Fat signaling, and, since they appear to come under the influence of Fz system in ds or fat mutants, they must also interface with the Fz system effector circuitry. Nevertheless, since the Fz and Fat receptors are so dissimilar from a molecular standpoint, their immediate effectors are likely to be quite distinct. Only by identifying the immediate and downstream effectors can it be understood how this polarized output occurs (Lawlor, 2013).

The situation is more complex as there are distinct polarization outputs to account for, just considering the Ds-Fat circuit. These will likely require distinct effector circuits. Studies in the Drosophila thorax, eye and wing already showed that asymmetric Ds and Fat accumulation leads to alterations of polarity evident as changes in the enrichment of the myosin Dachs. However, Dachs cannot be the sole effector in the embryonic epidermis as ABPs are correctly placed in its absence. Strong evidence for distinct Fat effectors also derives from elegant work showing that Fat affects junctional polarity, and is important for columnar cell alignment within the denticle field (Marcinkevicius, 2013). However, junctional polarity is most severely affected over a domain distinct from that exhibiting the most striking ABP placement defects (Marcinkevicius, 2013). And, while the role in junctional reorganization is most clearly defined among denticle field cells, it appears to apply across the smooth field also (Marcinkevicius, 2013). In contrast, the current study showed that polarization of ABPs can only occur over the denticle and not smooth field. In fact the role of Fat in alignment appears genetically separable from that in ABP placement (Marcinkevicius, 2013). Finally, different labs have identified distinct critical regions of the Fat intracellular domain necessary for polarity signaling. Thus, the mechanisms underlying polarity signaling through Ds- Fat await the identification of these different effector circuits (Lawlor, 2013).

EFFECTS OF MUTATION

Lethal mutations at the fat locus in Drosophila cause imaginal discs to continue to grow by cell proliferation far beyond their normal final size. During a greatly extended larval period, the overgrowing imaginal discs develop additional folds and lobes, but retain a single-layered epithelial structure. In the wing disc, the additional lobes originate in the proximal fold area, and in the extra tissue the cells are less columnar than normal. Mutant disc cells lack zonulae adherents as well as associated microtubules and microfilaments, and they show an abnormal distribution and reduced density of gap junctions. The effect on growth is disc-autonomous as shown by transplantation experiments. The overgrown imaginal discs retain the ability to differentiate adult cuticular structures, as shown by metamorphosis of discs after transplantation into wild-type larval hosts and by the ability of some mutant animals to develop to the pharate adult stage. The structures differentiated by mutant discs show many abnormalities including ingrowths, outgrowths, separated cuticular vesicles, and areas of reversed bristle polarity; some of these abnormalities suggest that the mutations interfere with cell adhesion as well as the control of cell proliferation. The fat locus is located in cytogenetic interval 24D5.6-7, and 18 alleles are known, including spontaneous, chemically induced, X-ray-induced, and dysgenic mutations (Bryant, 1988).

Recessive lethal mutations in the fat locus of Drosophila cause hyperplastic, tumor-like overgrowth of larval imaginal discs, defects in differentiation and morphogenesis, and death during the pupal stage. Clones of mutant cells induced by mitotic recombination demonstrate that the overgrowth phenotype is cell autonomous. Two recessive lethal alleles contain alterations in the fat coding sequence, and the dominant fat allele, Gull, contains an insertion of a transposable element in the 33rd cadherin domain. Thus, this novel member of the cadherin gene superfamily functions as a tumor suppressor gene and is required for correct morphogenesis (Mahoney, 1991).

The fat gene negatively controls cell proliferation in a cell autonomous manner. There are many ft alleles of different origin, most of which are pupal lethal, three are viable and one, ft^G (fat Gull) is a dominant antimorph. Bryant (1988) studied in some detail the phenotype of ft⁸ (previously known as fat floppy-disc, ft^fd) a pupal lethal with delayed entry in pupariation (8-9 days) and with abnormal-hyperplastic discs. Mutant ft⁸ pupae that survive to pharate adults show shorter and thicker leg joints, higher chaetae densities and deviant trichome and chaetae polarities. Wings are, however, of normal appearance. Delayed larvae show increasingly larger discs, with enlarged folds preferentially in the basal regions of the wing blade. However, transplants of these imaginal discs to metamorphosing wild-type hosts, did not show great departures from wild-type controls (Garoia, 2000).

Three different alleles: ft^G-rv, ft⁴ and a new one, isolated in a mutagenesis screen using P-element transposition and called l(2)79/18 have been examined in this study. l(2)79/18 was not associated with a P insertion and fails to complement other ft alleles; l(2)79/18 has been called ft¹⁸. ft^G-rv is a recessive lethal allele (Bryant, 1988) corresponding to the amorphic condition (Mahoney, 1991). Hemizygous ft^G-rv [ft^G-rv/Df(2L)sc19-1] larvae reach the pupal stage at 7-9 days and show hyperplastic discs (see Bryant, 1988). Homozygous ft⁴ larvae show a delay of 5-6 days until they reach pupariation and die as early pupae, prior to reaching the pharate stage. Their discs, in particular the wing disc, are many times larger than those of wild-type mature larvae, becoming larger the longer the larvae remain in culture. Homozygous ft¹⁸ larvae, in contrast, show less delay (up to 1-2 days) to pupariation, and 60% of the pupae reach the pharate stage. The pharate adults show abnormalities in cuticular patterns and structures in all appendages, similar to those of ft⁸ (Bryant, 1988). The adult wing of pharates is larger in size compared with the wild-type (1.5 times), with higher cell number (2.9 times) and higher trichome density (1.9 times). The shape of the wing is broad and short, enlarged in the A/P axis compared with controls. The basal wing region is more affected than distal regions, with vein abnormalities and abnormal trichome polarity. The chaetae pattern of the wing margin and the positioning of the vein sensillae are fairly normal, although chaetae are blunter and thicker (Garoia, 2000).

The two alleles ft⁴ and ft¹⁸ show different delays to pupariation. A comparison of sizes of the ft⁴ and ft¹⁸ wing discs from larvae of the same age reveals that ft¹⁸ grows faster than ft⁴. The sizes of ft⁴ and ft¹⁸ discs vary differently with the age of the larvae, suggesting a correlation between maximal disc wing size and delay to pupariation. Whether maximal wing disc size is allele specific was analyzed in flies carrying the ft⁴ and the ft¹⁸ mutations and a temperature-sensitive ecd¹ mutation. Homozygous ecd¹ larvae, when shifted from 20° to 29°C in the middle of third larval instar, fail to pupariate and survive as long as 3 weeks as larvae. Disc growth continues but levels off at maximal sizes after 20 days in ft⁴ ecd¹ and 15 days in ft¹⁸ ecd¹, whereas control ecd¹ wing discs stop growing at normal final size. These results indicate that extra growth is not indefinite and that different alleles reach different maximal sizes, being larger in ft⁴ than in ft¹⁸ discs. Similar results showing a maximal growth limit were reported by Bryant (1988) comparing 9-day-old discs of ft⁸ larvae with ft⁸ discs cultured in adult hosts for 21 days (Garoia, 2000).

The heteroallelic combinations ft⁴/ft¹⁸ and ft^G-rv/ft¹⁸ show a similar delay in pupariation (up to 1-2 days) and have a maximal disc size similar to that of ft¹⁸ homozygous. Both combinations reach the pharate stage with extreme ft phenotypes. Wing discs of ft^G-rv/ft⁴ combination are larger, larvae pupate at 7-9 days and die as pupae. In heteroallelic combinations with the deficiency for the locus, ft¹⁸ and ft^G-rv pupate at the same time and reach similar disc sizes, but ft⁴ pupates later and the discs reach larger sizes. Thus, delayed pupariation as in ft⁴ homozygous larvae or in discs maintained in non pupating hosts (ecd^-) allows for disc extra growth but only up to a maximal size. Combinations of the same ft alleles (and ft^G-rv; Bryant, 1988) with the weak viable allele ft⁺ are viable and of ft⁺ phenotype (Garoia, 2000).

The ft pharate adults (and mutant mosaic patches) show smaller cell surfaces or more density of trichomes. This effect on cell differentiation could correspond to smaller cell sizes during cell proliferation. Cell sizes of mature four days wing discs ft⁴ and ft¹⁸ were compared with those of wild-type. Cell densities in ft⁴ and ft¹⁸ discs are similar, and higher (1.2 times) than in wild-type discs. Faster growth rate and smaller cell size could be related to cluster size of cells in different cell-cycle stages. The wildtype stg expression that marks the G2/M transition, occurs in synchronic clusters of 5.1 +/- 2 cells throughout larval development. The sizes of stg clusters in ft¹⁸ and ft⁴ discs yield values in ft⁴ and in ft¹⁸ that are not different from those of wild-type discs (Garoia, 2000).

The study of cell behavior of lethal allelic conditions can be carried out in genetic mosaics. This allows for a study of phenotypes of mutant territories but also of their relationships with neighboring wild-type territories. Morphogenetic mosaics have been studied in large M⁺/ M⁺ (ft homozygous) clones, initiated in a M;ft heterozygous background as well as in clones of ft/ft and ft⁺/ft⁺ twin cells. The analysis of M⁺/M⁺ clones initiated at 60 +/- 12-h AEL associated with homozygosis of ft^G-rv, ft¹⁸ and ft⁴ reveals several features related with ft cell proliferation and differentiation. A fraction of M⁺ clones initiated at this age cross the D/V boundary and most clones tend to fill one or several intervein sectors. In wild-type wings the corresponding figures are 13.8% and clone sizes of 1.1 sectors on average. The corresponding figures for ft^G-rv are 25% and 1.3; 35.2% and 1.2 for ft¹⁸, and 19.2% and 0.8 for ft⁴, reflecting early growth advantages of mutant clones. In contrast with wild-type controls, ft clones tend to fill proximal regions of the wing blade without reaching distal regions of the wing. The distal borders of ft clones are rounded, i.e. cells grow there perpendicular to the A/P boundary. In addition, they are smooth in contrast with the indented borders found in M⁺ control clones. In all of these features, ft^G-rv and ft¹⁸ are more extreme than ft⁴ (Garoia, 2000).

ft clones cause outgrowths in proximal but not in distal wing regions. There are blisters of variable sizes that evaginate from the wing surface, and have irregular trichome polarities, usually perpendicular to the clone border. Trichome density (cell size) in clones (with or without outgrowths) is higher than in wild-type controls, 1.7 times in ft^G-rv, 2.2 times in ft¹⁸ and 1.4 in ft⁴ clones, indicating that mutant cell size is smaller than in wild-type. Mutant clones appearing between veins enlarge the corresponding sectors, with more cells than in control mosaics (ft^G-rv 1.4 times, ft¹⁸ 1.4 and ft⁴ 1.3). Mutant territories that overlap veins differentiate thicker than normal vein ribbons. In the wing margin ft M⁺ clones cause also higher density of chaetae. The ft chaetae (M⁺/M⁺) are smaller than the M/M⁺ heterozygous chaetae, despite not being M^- -- that is, they are not larger but rather are shorter and blunter. In these features ft^G-rv and ft¹⁸ are more extreme than ft⁴. In the notum, ft M⁺ clones are large, with more cells than control M⁺ clones, leading to an increase in the total notum surface. They contain many more chaetae (1.6 2 times in ft^G-rv, 1.7 in both ft¹⁸ and ft⁴), and higher cell (trichome) density (1.4 times in ft^G-rv, 1.4 in ft¹⁸ and 1.2 in ft⁴). Similar pattern deviations occur in the legs and head capsule (see Bryant, 1988). Tergite clones, however, show normal patterns of pigment, chaetae and trichomes (Garoia, 2000). Based on these features of clone size, trichome and chaetae density and chaetae size, the ft^G-rv and ft¹⁸ alleles appear again more extreme than the ft⁴ allele (Garoia, 2000).

The autonomous extra cell proliferation found in M⁺ mosaics can be quantitatively ascertained in a comparison of ft and ft⁺ homozygous cells in a twin test. In this test ft cells, either ft^G-rv, ft¹⁸ or ft⁴, are labelled with forked and the twin controls with crinkled (ck). Clones were initiated at 60 +/- 12 h AEL. The size (in cell number) of ft mutant clones was compared that of their ck twins. ft^G-rv and ft¹⁸ clones are much larger (average ratio of 6.5 and 14.9, respectively) than twin controls. The same holds for ft⁴ clones, although the ratio is only 3.0. Trichome density in these small clones is also like that in large M⁺ clones (1.7 in ft^G-rv, 1.7 in ft¹⁸ and 1.4 in ft⁴). This phenotype is cell autonomous because it appears even in small (late initiated) clones. As in Minute mosaics, clones that include two adjacent veins give rise to larger outgrowths, than those that include only one vein. These clones have increased cell density and deviant cell polarity, independently of clone size. ft⁴ clones are smaller than ft¹⁸ and ft^G-rv, and therefore fewer clones of this genotype overlap two adjacent veins (in these few cases they correspond to the largest clones). The cell viability of ft clones is like that of wildtype cells because the frequency (33% for ft^G-rv, 31% for ft¹⁸, 21% for ft⁴) off clones without ck twins corresponds to that of controls (27%) and to the distance of the Dp f⁺ to ck on the mitotic map (Garoia, 2000).

As in large M⁺ clones, borders of small ft clones in twins are smooth and in some cases they also show outgrowths evaginating from the wing surface. It is interesting to notice here that large and small ft clones do not merely abut adjacent veins, as control clones do, but very frequently cross over the veins and stop a few cells beyond the territory differentiating vein histotype running parallel to them. The frequency of vein overlapping clones is 64.7% for ft^G-rv, 62.5% for ft¹⁸ and 67.5% for ft⁴ clones compared respectively to 25, 23 and 27.5% for twin ck clones. The observation that M⁺ ft cells grow preferentially in proximal regions, avoiding the distal wing, is also reflected in a twin test. The topological location of the mother cell of the twin in the anlage can be ascertained in the adult mosaics as the point where the two members of the twin come into contact. The mutant cells preferentially grow towards the base of the wing, while their twins grow towards more distal regions (that holds for ft¹⁸ and ft^G-rv and less so for ft⁴). This at least accounts for 80% in ft¹⁸, 71% in ft^G-rv and 61% in ft⁴; exceptions to the rule appear in the proximal and most anterior wing regions. This effect is evident in the frequent separation of the two members of the twin (18% in ft¹⁸). Abnormal cell differentiation in vein thickening, chaetae density and chaetae morphogenesis in these small clones is, as expected, like that in large M⁺ clones (Garoia, 2000).

The abnormal behavior of ft mutant cells in cell proliferation, as reflected in abnormal disc sizes and shapes prompted an analysis of possible genetic interactions with mutations in other genes that affect these parameters. Such interactions can be studied in doubly mutant combinations, either in the morphology of imaginal discs of doubly mutant lethal larvae, or in genetic mosaics. Since ft mutant discs become increasingly more abnormal with the age of the larva, comparisons of phenotypes of the mutant combinations are difficult. The selector gene of the dorsal compartment, ap, causes in the mutant condition (ap^rK568) a major reduction of the wing pouch. The double homozygote ft¹⁸ aP^rK568 is lethal (L1-2 phenoeffective phase), suggesting early synergetic effects, possibly in organs other than imaginal discs. The nub² allele of the nubbin gene causes smaller wing disc and a strong reduction of growth in the proximodistal axis of the wing. Surprisingly the doubly mutant combination ft¹⁸;nub² causes a strong delay in pupation (from 2 to 5 days), allowing the wing discs to be considerably enlarged. These effects are apparently synergetic because 5 days old discs are larger than ft¹⁸ controls. The extreme folding of 7-10 day old discs indicates overgrowth of the wing pouch despite being mutant for nub²; this could simply reflect epistasis of ft¹⁸ over nub (Garoia, 2000).

The Drosophila fat gene is exclusively expressed in ectodermal derivatives, both in the embryo and imaginal tissues (Mahoney, 1991) and this paper shows that ft is transcribed in all imaginal discs. The Fat protein 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; Ponassi, 1999). All these domains appear in the FAT cadherin homolog of vertebrates, except for its cytoplasmic domain (Ponassi, 1999). Classic vertebrate cadherins contain only the cadherin domains. A Drosophila homolog to these cadherins is the dachsous gene. It is not known how the Drosophila ft gene functions in cell adhesion, but the presence of EGF-like repeats in other C-terminal regions also suggests a role in cell-cell signaling, as a receptor or as a ligand. The molecular description of some mutations of ft ( ft¹⁰, ft¹⁵, ft^G and ft^G-rv) shows that they encode truncated proteins containing different fractions of the cadherin region but lacking the EGF domains and more proximal domains. A ft^G revertant ( ft^G-rv), resulting from a complex rearrangement within the ft gene, may correspond to a ft null allele (Mahoney, 1991). All the lethal alleles are phenotypically similar. They could correspond to functional nulls, if the cadherins cannot be anchored to the cell membrane and lack the putative signaling regions, but the remaining secreted cadherin domains could act as dominant negative competitors of normal cadherins. This is the explanation given for ft^G (Mahoney, 1991). Developmental genetic studies have been carried out in four alleles ( ft⁸, Bryant, 1988; ft^G-rv, ft⁴ and ft¹⁸; Garioa, 2000). Of these alleles only the molecular nature of the ft^G-rv allele is known (Mahoney, 1991). ft⁴ and ft¹⁸ are transcribed, but the signal is weak and appears only in the tip of the wing blade. This low expression could mean that ft transcription is dependent on normal ft activity (Garoia, 2000).

The phenotypic consequences of ft alleles are multiple: (1) most alleles are pupal lethals; (2) imaginal discs reach large sizes, with abnormally convoluted folds; (3) pupariation is delayed; (4) proliferating embryonic and adult cells are smaller than wild-type cells; and (5) differentiation of trichomes and chaetae is affected, not only in terms of their size and polarity, but also in their numbers and patterning (Bryant, 1988 and Garoia, 2000). Interestingly, hyperplasia is associated with an abnormal pattern of gene expression, as visualized in two-dimensional protein gels of ft⁴ discs. They show 6-8 fewer spots than wild-type; 11 abundant polypeptides are either new or absent and another 11 show significant quantitative differences with wild-type (Fernandez-Santaren, 1998). The three alleles studied in this work differ in several respects: ft^G-rv hemizygotes ft¹⁸ homozygotes and heteroallelic combinations show maximal delays to pupariation of 1-2 days, whereas ft⁴ larvae may grow for a further 4 days before pupariation. Experiments in ecd¹ permissive larval environment, in which pupariation is delayed, however, indicate that ft mutant discs may continue growing but only up to a maximal size. The ft¹⁸ and ft^G-rv alleles show higher growth rates and more extreme phenotypes than homozygous ft⁴ in mosaics. Thus the ft⁴ allele may be more hypomorphic than the others, but by itself causes delays in pupariation which allow discs to grow larger than in the other alleles that pupate earlier (Garoia, 2000).

How could these mutational alterations of ft account for the complex syndrome of mutant phenotypes at the cellular level? Clonal mutant phenotypes allow for study of developmental parameters of the extreme alleles. Mosaic patches (M⁺ and twins) of the three alleles show overgrown or enlarged territories with more, and smaller, cells than controls. The autonomous extra growth in mosaics occurs everywhere in all the wing disc derivatives examined, within an otherwise normal pattern. In proximal wing regions these extra growths may lead to large bulges. These clones, however, respect the compartmental and vein restrictions. Mosaic extra growth is locally restricted to the clones, the surrounding wild-type territories retaining their wild-type aspect and sizes without signs of positional accommodation. These findings indicate that the mutant territories are simply modifying some proliferation parameters of an otherwise normal, allometrically growing wing disc (Garoia, 2000).

The abnormal cell behavior in clones suggests failures in cell adhesiveness and cell recognition. Thus, ft mutants clones contain trichomes with abnormal polarities, mainly pointing to clone borders and have smooth borders indicating incompatibilities with wild-type cells. In distal wing regions, clone borders are perpendicular to the proximo-distal axis and not parallel, as in controls, indicating preferential allocation of post-mitotic cells to proximal wing positions. This is more manifest in twin clones where the mutant partner grows proximally. In contrast, mutant territories show abnormal cell differentiation patterns, including thicker or wider veins and patterns with more chaetae at higher density, suggesting a role of ft in patterned cell differentiation. The same holds for the territorial expression of wg and dpp expanded to wider territories in mutant discs as compared with wild-type. Thus, whereas the mutant features of trichome polarity and smooth clonal borders can be directly explained by adhesiveness failures, the wider differentiation patterns of chaetae and veins could reveal failures in cell communication in the subdivision of pre-pattern territories. The latter effects are possibly not mediated by failures in lateral inhibition because ft and N or Ax double combinations do not increase the phenotypes beyond mere additivity. ft mutant alleles also affect the differentiation of chaetae; they are shorter and have blunter shafts, suggesting an additional role of ft also in final cell differentiation, possibly related to cytoskeleton anomalies observed in EM pictures of imaginal disc cells (Bryant, 1988; Garoia, 2000).

Overgrowth and abnormal cell behavior of ft cells during proliferation seem to be related to failures in cell signaling and/or cell adhesion. The normal expression patterns of genes with regional specificity, such as ap, en, wg and dpp shown in large ft discs indicate that the overgrowth of the discs is allometric, i.e., not associated with duplications or abnormal local regeneration. This is also the case for other hyperplastic discs, like lethal giant discs (lgd) whose discs show ectopic expression of dpp and wg. Overgrowth of mutant discs in delayed pupariating hosts (ecd^- mutant background) reaching a maximal size, indicates again that overgrowth is allometric, i.e. normal cell proliferation parameters are maintained. Thus, enlarged territories in clones included between normal restriction boundaries (compartments and veins), could be due to failures in cell-cell signaling needed to intercalate positional values defined by borders of clonal restrictions (Garoia, 2000).

Genes that are required for cell proliferation control in Drosophila imaginal discs were tested for function in the female germ-line and follicle cells. Chimeras and mosaics were produced in which developing oocytes and nurse cells were mutant at one of five imaginal disc overgrowth loci (fat, lgd, lgl, c43 and dco) while the enveloping follicle cells were normal. The chimeras were produced by transplantation of pole cells and the mosaics were produced by X-ray-induced mitotic recombination using the dominant female-sterile technique. The results show that each of the genes tested plays an essential role in the development or function of the female germ line. The fat, lgl and c43 homozygous germ-line clones fail to produce eggs, indicating a germ-line requirement for the corresponding genes. Perdurance of the fat plus gene product in mitotic recombination clones allows the formation of a few infertile eggs from fat homozygous germ-line cells. The lgd homozygous germ-line clones give rise to a few eggs with abnormal chorionic appendages, a defect thought to result from defective cell communication between the mutant germ-line and the nonmutant follicle cells. One allele of dco prevents egg development when homozygous in the germ line, whereas another allele has no effect on germ-line development. Fs(2)Ugra, a recently described follicle cell-dependent dominant female-sterile mutation, allows the analysis of egg primordia in which fat, lgd or lgl homozygous mutant follicle cells surround normal oocytes. The results show that the fat and lgd genes are not required for follicle cell functions, while absence of lgl function in follicles prevents egg development (Szabad, 1991).

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

High-resolution two-dimensional (2D) gel electrophoresis coupled with computer analysis has been used to construct a quantitative protein database of Drosophila mature wing imaginal discs. The level of expression for all of the detected proteins has been quantitatively determined. This database has been used to evaluate changes in the patterns of protein synthesis in wing imaginal discs from two Drosophila melanogaster mutants with abnormal wing disc development: fat and two different alleles of lethal (2) giant disc. Patterns of pulse-labeled proteins of the different mutants show variations in both qualitative and quantitative parameters of synthesis. In this comparison, specific sets of protein changes characteristic of both alleles of the same locus and a set of protein changes common to both loci have been detected. How the abnormal expression of these proteins relates to the abnormal process of mutant hyperplasia is discussed (Santaren, 1998).

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

Developmental compartments and planar polarity in Drosophila

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 have not found any difference in behavior between the different compartments. This study shows that the three genes, four-jointed, dachsous, and fat, cause opposite effects in anterior and posterior compartments. For example, in anterior compartments, clones ectopically expressing four-jointed reverse the polarity of cells in front of the clone, while, in posterior compartments, they reverse behind the clone. These three genes have been reported by others to be functionally linked. This discovery impacts on models of how cells read polarity. At the heart of one class of models is the hypothesis that cell polarity is determined by the vector of a morphogen gradient. Evidence is presented that cell polarity in the abdomen depends on at least two protein gradients (Fj and Ds), each of which is reflected at compartment borders. Consequently, these gradients have opposing slopes in the two compartments. Because all polarized structures made by abdominal cells point posteriorly, it is surmised that cells in each compartment are programmed to interpret these protein gradients with opposite signs, pointing up the gradient in one compartment and down the gradient in the other (Casal, 2002).

Two other genes resemble fj with regard to compartment-specific effects: dachsous (ds) and fat (ft). In both cases, UAS transgenes cannot be easily made, so only the effects of removing the genes have been studied. 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).

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

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

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).

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

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

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).

Drosophila genetic variants that change cell size and rate of proliferation affect cell communication and hence patterning

The role of genetic variants that affect cell size and proliferation in the determination of organ size has been investigated. Genetic mosaics of loss or gain of function were used in six different loci, which promoted smaller or larger than normal cells, associated with either smaller or larger than normal territories. These variants have autonomous effects on patterning and growth in mutant territories. However, there is no correlation between cell size or rate of proliferation on the size of the mutant territory. In addition, these mosaics show non-autonomous effects on surrounding wild-type cells, consisting always in a reduction in the number of non-mutant cells. In all mutant conditions the final size (and shape) of the wing is different from normal. The phenotypes of the same variants include higher density of chaetae in the notum. These autonomous and non-autonomous effects suggest that the control of size in the wing is the result of local cell communication defining canonic distances between cells in a positional-values landscape (Resino, 2004).

Size of insect organs is sex- and species-specific. In the Drosophila wing, where most of the studies on size control have been carried out, the determination of the size of imaginal disc is disc-autonomous. Young imaginal discs transplanted to the abdomens of adult flies grow after several days of culture, irrespective of hormonal and nutritional conditions, to a maximal size that corresponds to that of mature imaginal discs. Minute mosaics and regeneration experiments reveal that a final normal size is attained irrespective of the rate of cell proliferation. Clonal analysis of cell proliferation in wild-type wings show regional differences related to specification or differentiation, indicative of local as opposed to global control of organ size. Size of the growing imaginal disc depends on the allocation of postmitotic cells along the main axes of the wing in regimes that change with developmental time. There is no indication that cell proliferation or cell allocation relates to the position of cells with respect to distances to compartments boundaries, where postulated diffusible morphogens are at maximal concentration (Resino, 2004).

If control of cell proliferation is local, the question arises as to how this is achieved. Can variations in cell size affect the final size of the organ or its proliferation parameters? These variations can be produced using mutations, usually lethal in organisms, and have to be studied in genetic mosaics. Mosaics of haploid territories (with half the cell size of diploid cells) led to bigger territories with more cells than diploid territories. Male wings have less and smaller cells than females, characteristics that are locally autonomous in gynandromorphs. For mutations that affect cell size, it has to be considered that they cause different perturbations that may affect other cellular parameters in addition, such as cell viability, proliferation rate or cell adhesion, which make difficult the interpretation of the phenotype. Thus, the insufficient function of genes involved in cell cycle progression, such as string (stg), cdc2 and cyclins or E2F (cycE positive regulator), may retard the cell cycle and cause cell mortality, an increase in cell size and smaller mosaic territories in otherwise apparently normal sized discs. Mutant cells in these mosaics do not differentiate properly. On the contrary, over-expression of the same cell cycle genes (i.e. stg, cycE, cycD-cdk4) or of their activators (i.e., E2F) in imaginal disc clones cause acceleration of their characteristic phases of the cell cycle, as well as a reduction of cell size (except cycD-cdk4 combination) and an increase in number of cells of the mutant territory compared with control cells in apparently normal sized mosaic wing discs. These effects are more extreme in some genetic combinations (e.g., cycE-stg) because they cause an acceleration of the whole cell cycle. These studies conclude that cell size reduction/increase is 'compensated' by increment/decrement in cell number in the mutant territory, as if the organ would compute a global normal size, because the mutant wing disc territories have an apparent wild-tupe size. This interpretation is biased by the fact that those mosaics show high cell mortality. When this is prevented with the coexpression of P35, the extra growth of the mutant territories in discs and clones is even higher, leading to abnormally shaped mutant territories. The over-expression of the cycD-cdk4 combination in the eye reaches the adult stage and causes larger and abnormally shaped mutant territories. These studies have not analyzed non-autonomous effects in non-mutant territories of the same discs (Resino, 2004 and references therein).

Less drastic mutant effects associated with cell viability are obtained with mutant perturbations in the signal transduction and reception of the insulin pathway. As a rule, loss of function of Drosophila Insulin Receptor (Inr), chico or Dp110 causes reduction in both cell size and cell number of mutant territories. This is similar to what happens in wild-tupe flies exposed to malnutrition or premature metamorphosis. This holds for each member of the insulin receptor pathway except for Drosophila S6 kinase (S6K), because S6K loss of function only reduces cell size but not cell number. On the contrary, the gain of function of genes of this pathway causes larger cells and an increase in the number of cells of the mutant territory in mosaics. The loss of function of myc in diminutive mutants leads to smaller flies, with smaller cells, in addition to poor cell viability. Its overexpression causes larger cells but not larger territories, suggesting that in this latter condition (but not the former) the wing size in globally controlled by a normalizing compensating mechanism (Resino, 2004 and references therein).

The results show a great heterogeneity in the response of regional size to genetic perturbations that cause variations in cell size during cell proliferation. In fact, both smaller or larger than normal cell size may accompany normal, larger or smaller mutant territories. In the present paper, the effects on cell proliferation of mutant conditions in six loci that cause smaller and larger cell sizes have been studied. Of these, one corresponds to a new gene and five to previously studied genes that affect cell size. They were chosen as examples of the cell behavior variants, as representatives of mutant effects on cell size (larger and smaller than normal) and rate of proliferation (slower and faster than normal). The choice was made without considering the genetic/molecular bases of the corresponding wild-tupe alleles, in any case mechanistically far separated from the analyzed phenotype. Their autonomous effects in mutant territories and in the mosaic wing as a whole were studied: nonautonomous effects were documented as well (Resino, 2004).

Adult cell size is measured by the exposed planar surface of the cuticle cells. In principle, this may not reflect the size of the proliferating cells, when organ size is determined. However, in some of the cases examined in this study, cell dissociation has revealed by direct estimation the larger or reduced cell size in the proliferating wing disc cells. In others, cell size during growth is inferred by the mutant effects on pattern formation, a process that precedes final cell differentiation, as in the notum pattern of microchaetae. This pattern results from the singularization of sensory organ mother cells (SOMC) in a field of epidermal cells through a process of lateral inhibition in a field of proneural clusters. Thus, the final pattern reveals cell-cell interactions or communication, as observed in the form of cell projections emanating from epidermal cells. It holds for all mutant and genetic combinations examined in this study that the pattern, number and density of chaetae are all altered in the notum (in the mutant Dmcdc2^E1-24 cells fail to differentiate chaetae). In all cases, chaetae appear more densely spaced (separated by less epidermal cells) associated with an increase in the total number of chaetae. These variations to the wild-tupe condition suggest that mutant cells have impaired the capacity to signal among themselves to define spaced SOMC singularization. Whether this is or is not associated with cell size in individual cases is not known. These pattern effects reveal abnormal cell communication between cells during cell proliferation (Resino, 2004).

Although less easy to measure in mosaic nota, there is a phenotypic association of variable cell size with a reduction (in l(3)Me10, gig^Me109, Dp110^D945A) or an increase (EP(3)3622, ft^a13, Dp110-CAAX) in notum sizes. But there is no apparent causal relation between both parameters of cell size and number of cells making the adult notum. Perhaps cell viability associated with the mutation, as in l(3)Me10 and gig^Me109, may account for the observed lack of correlation between both parameters. However, these effects on notum size in other cases may also reflect failures in cell-cell communication leading to more or less cell proliferation (Resino, 2004).

The relationship between cell size and growth can be more readily measured in the wing. The studied genetic variants can be grouped, based on variations in these parameters, as follows:

• (1) Reduction in cell size and in number of cells within a wing sector is associated with a reduction of the size of the mosaic sector (l(3)Me10 and Dp110D954A). This is a common phenotype in many mutant conditions such as in mosaics of vein and torpedo, both of the Epidermal growth factor receptor (EGFR) pathway. This situation is very common to many loss of function alleles of many genes (Resino, 2004).
• (2) Reduction in cell size and increase in number of cells is associated with increase in the sector area (EP(3)3622 and ft^a13) (Resino, 2004).
• (3) Increase in cell size and reduction in number of cells in mosaic sectors is associated with a reduction in the sector area in gig^Me109 and Dmcdc2^E1-24 (Resino, 2004).
• (4) Increase in cell size and increase in cell number is associated with an increase in the mosaic sector area in Dp110-CAAX (Resino, 2004).

The autonomous effects on reduced clone size can result from the poor viability of mutant cells (l(3)Me10 or Dmcdc2^E1-24), as shown in twin clonal analysis and cell death monitoring. The increased clone size of EP(3)3622, ft^a13 or Dp110-CAAX reflects higher than normal cell proliferation, however there are no correlations between cell size and clone size. Despite this lack of correlation it holds for all mutants examined in this study that, concerning the non-autonomous effects on growth in the mosaic wing sector: the non-mutant cells of the sector are always reduced in number. No cases were found in which the reduction or increase in sector size by the presence of mutant territories is compensated by wild-tupe cells to obtain a normal sized sector (Resino, 2004).

The mosaic wings show, in addition to autonomous effects within mutant sectors, non-autonomous effects in the rest of the wing. It holds for all cases studied that wings with entire or mosaic wing sectors show a reduction in the total area of the wing or more in particular in non-mosaic areas (sectors or compartments) of the wing. This phenomenon is designated as 'positive' or 'negative' accommodation, depending on its correlation with the size of the mutant region. This phenomenon could be easily trivialized for mutations that cause size reduction and 'positive accommodation'. It is arguable that there are not enough cells in the mutant territories to confront with normal growing cells abutting the clone, the sector or the mutant compartment. 'Positive accommodation' could result from adjustment between poorly growing cells and normal ones. However this large effect hardly explain 'negative accommodation' for the whole wing. 'Negative accommodation' occurs in mosaic wings with mutant territories with more cells than normal, such as EP(3)3622, ft^a13 or Dp110-CAAX (Resino, 2004).

Reduction in the size of non-mutant territories in mosaic wings cannot be explained either by delay in development (mosaic flies hatch at the same time as sib controls) or age of clone initiation. It cannot be explained either by cell death, because there is enough time for extraproliferation to reach normal sized wings, since it occurs in mosaics where cell death has been massively induced in Gal4 territories. 'Negative accommodation' is surprising because one would expect that larger than normal mutant territories should provide adjacent wild-tupe cells with more growth signals (Resino, 2004).

To account for this 'negative accommodation' it is postulated that mutant cells do not convey among themselves and to wild-tupe cells sufficient signals necessary for them to proliferate. These signals may depend on cell-cell communication. In the notum it has been seen that failures in cell-cell communication may account for abnormal chaetae patterning and notum size. The same may apply to the wing blade, although there are not enough pattern elements to support this inference (Resino, 2004).

A model has been proposed to explain controlled cell proliferation, based on local cell-cell signalling, as opposite to reception of graded amounts of morphogens emanating from compartment boundaries, such as Dpp and Hedgehog or Wingless. The Entelechia model (Interactive Fly editor's note: 'Entelechia' is a Greek term coined by Aristotle for the complete reality or perfection of a thing, and refers to the process of coming into being) states that cell proliferation results from local interactions between neighboring cells. In these interactions, cells compute positional values, presumably expressed in the cell membrane. Positional value discrepancies elicit cell division and readjustment of positional values of daughter cells to those of neighboring cells. These values differ along the two main axes of the wing, A/P and Pr/Ds. Cell proliferation occurs within clonal boundaries; those of compartments in the early disc and other boundaries, such as veins, later. In these boundaries the interchange of some type of signals help to increase positional values at the border, eliciting cell division, cascading down to intermediate regions with minimal values. Cell proliferation is intercalar and driven by differences in positional values between cells with lower and higher values. These minimal differences may reflect canonic efficiencies ('increments') in transduction of signals (ligands/receptors) between neighboring cells. Cell division ceases in the anlage when cells in the boundaries reach maximal values and their increments, between all the cells of a region become minimal. The anlage has then reached the Entelechia condition of growth, characteristic of the organ, the sex and species (Resino, 2004).

An organ such as the wing, grows co-ordinately through compartments and clonal boundaries because maximal positional values result from cell interactions at both sides of the boundaries. In this respect compartments or wing sectors are not independent units of cell proliferation. This was first seen in bithorax-Complex (bx-C) mutants, where either the A or P compartments of the haltere were transformed to A or P compartments of the wing. The untransformed A or P haltere compartments contain now more cells, and the transformed ones less than a wild-tupe A or P wing compartment. This accommodation is explained as due to the reduced extent of the compartment boundary between apposed mutant and nonmutant compartments. Similar accommodation effects have been already reported in other mutant conditions, such as mutants of the EGFR pathway in extramacrochaetae (emc) and in nubbin (nub). In the latter case, the presence of proximal wing mutant territories causes a distal reduction in growth in all the wing compartments (Resino, 2004).

The Entelechia model helps to understand the behavior of mosaic wings for the mutants examined in this study. In all cases, clones or regions with smaller or larger cells and with less or more cells than normal, cause autonomous effects on growth in mutant territories but also a non-autonomous 'accommodation' in the rest of the wing formed by wild-tupe cells. It should be emphasized that the effects on proliferation between mutant and non-mutant territories are reciprocal; the non-mutant territories rescuing proliferation in the mutant territories and vice versa. It is hypothesized that failures in cell communication of positional values to/from neighboring mutant or non-mutant cells affect the 'increment' values of the model. This leads to reduced proliferation in both genetic territories between cells because cells cannot generate higher positional values and thus promote intercalar proliferation. This finding indicates that the size of territories does not depend on distances from diffusible morphogen sources, measured either in physical terms or in number of cells, or on other postulated parameters such as measuring global cell mass or wing length. How would these global dimensions be defined, and how would they be computed by individual cells? How would one explain that mosaic territories separated from compartment boundaries (or morphogen sources) can affect the growth of wild-tupe territories far away all over the wing? It seems rather that cell proliferation control depends on local cell interactions (cell-cell communication) that define positional values throughout the whole growing organ (Resino, 2004).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

However, this study has shown that misexpressed Ft retains PCP activity in a ds mutant that eliminates detectable cell surface Ds. Thus, Ft activity is apparently not strictly dependent on patterned Ds expression. Again, this is consistent with the greater severity of ft mutant phenotypes compared with ds, and with the finding that uniform misexpression of ft but not ds can cause PCP defects. Since there is no evidence for homophilic Ft binding, the unbound Ft molecule may have basal PCP activity. Alternatively, low-level homophilic binding or heterophilic binding to some unknown ligand may activate Ft in the absence of Ds (Matakatsu, 2004),

It is not yet known how Ft-Ds interactions regulate the polarized redistribution of the core polarity proteins in the older pupal wing. The cytoplasmic domains of Ft and Ds contain potential regions for ß-catenin binding, and ft and ds mutants can enhance the effects of ß-catenin (Armadillo) misexpression. However, although expression of DE-cadherin in vitro results in a detectable concentration of Armadillo at the cell membrane, no similar effects were detected after expression of ft or ds. Moreover, clones homozygous for a strong armadillo mutation do not affect PCP. It has also been suggested that the cytoplasmic domain of Ft binds to and changes the activity of Grunge, the Drosophila homolog of the Atrophin transcriptional co-repressor, but it is not known whether this interaction is altered by Ft-Ds binding (Matakatsu, 2004),

The studies examining the timing of Ds activity suggest that its effects on the polarization of the core polarity proteins are likely to be indirect, since Ds acts before the polarized redistribution of the core polarity proteins within cells can be detected. Patterned misexpression of Ds at later stages, during the time of core protein polarization, has no effect on PCP. The period sensitive to ds misexpression is roughly congruent with the period of early Fz activity; if loss of Fz is limited to a period from 6 to 24 hours AP it leads to distinct, ds-like PCP defects. Thus, early Fz and Ds activity may be linked, or they may share a common target (Matakatsu, 2004),

The only known sign of cell polarization during the stages sensitive to Ds and early Fz activity is the redistribution of the Widerborst PP2A regulatory subunit from the anterior-proximal side to the distal side of wing cells at some time between 8 and 18 hours AP. Reductions in Widerborst activity can disrupt the polarized redistribution of Fmi and Dsh, suggesting an instructive role. However, Widerborst polarization is not affected by ectopic Fz expression, making it less likely that Widerborst polarization mediates early Fz activity (Matakatsu, 2004 and references therein),

A final interesting feature of the results is the preferentially heterophilic binding observed between Ft and Ds in vitro. This result is consistent with analyses of protein distribution within and adjacent to ft and ds mutant and overexpression clones. With the exception of the desmosomal cadherins, this kind of binding is unusual for cadherin-like proteins (Matakatsu, 2004),

A number of mammalian Fat-like (Fat1, Fat2, Fat3, XP_227060) and Ds-like (Protocadherin 16, Cdh23) proteins have been identified. Mutations and knockouts have been examined for a few of these; however, conjectures about the bases of the mutant phenotypes have largely assumed that these proteins mediate homophilic cell adhesion. It will be interesting to see whether the preferentially heterophilic interactions observed in Drosophila are preserved in similar mammalian proteins (Matakatsu, 2004),

The tumor suppressor gene fat modulates the EGFR-mediated proliferation control in the imaginal tissues of Drosophila melanogaster

Molecules involved in cell adhesion can regulate both early signal transduction events, triggered by soluble factors, and downstream events involved in cell cycle progression. Correct integration of these signals allows appropriate cellular growth, differentiation and ultimately tissue morphogenesis, but incorrect interpretation contributes to pathologies such as tumor growth. The Fat cadherin is a tumor suppressor protein required in Drosophila for epithelial morphogenesis, proliferation control and epithelial planar polarization, and its loss results in a hyperplastic growth of imaginal tissues. While several molecular events have been characterized through which fat participates in the establishment of the epithelial planar polarity, little is known about mechanisms underlying fat-mediated control of cell proliferation. Evidence is provided that fat specifically cooperates with the epidermal growth factor receptor (EGFR) pathway in controlling cell proliferation in developing imaginal epithelia. Hyperplastic larval and adult fat structures indeed undergo an amazing, synergistic enlargement following to EGFR oversignalling. Such a strong functional interaction occurs downstream of MAPK activation through the transcriptional regulation of genes involved in the EGFR nuclear signalling. Considering that fat mutation shows di per se a hyperplastic phenotype, a model is suggested in which fat acts in parallel to EGFR pathway in transducing different cell communication signals; furthermore its function is requested downstream of MAPK for a correct rendering of the growth signals converging to the epidermal growth factor receptor (Garoia, 2005).

These results support the hypothesis of a relevant functional interaction between ft and genes of the EGFR pathway. When EGFR signalling is raised in fact, an amazing, non-additive increase in ft-induced proliferation is observed. ft clones in UAS-rho or UAS-raf^GOF wings where an EGFR oversignalling is induced show the same distinctive features of those induced in a wild-type background (tissue hyperplasia, reduced cell size, loss of planar polarity), with a phenotype much more severe concerning outgrowth number and dimensions. On the contrary, in experiments where the EGFR signalling is reduced, ft-induced hyperproliferation results are partially suppressed. The same trend is observed in the adult eye; the ft head-capsule where rho or raf^GOF were ectopically expressed was particularly enlarged (Garoia, 2005).

The most dramatic effects were however observed in the eye and wing imaginal discs where the EGFR signalling was increased in the presumptive dpp expression domains. The controls did not show significant phenotypes; conversely, in ft discs severe effects were observed, including non-autonomous aberrations in the disc morphology. In the ft raf^DN eye disc a strong non-autonomous reduction of the eye presumptive territory was observed that may be caused by decrease of proliferation rate and/or by increase of cell death. Non-autonomous cell death may result from cell competition, ectopic cell proliferation or experimentally induced apoptosis. An increase in non-autonomous cell death was observed not only in ft raf^DN discs but in all the ft/UAS combinations analyzed. Thus, cell death does not seem to be the mechanism through which the growth deficit occurs in the ft raf^DN eye disc, suggesting a more complex interaction between ft and EGFR activity in the modulation of eye disc growth (Garoia, 2005).

Therefore ft interacts with the EGFR pathway modulating its proliferative signal; ft function is therefore involved ingrowth regulation, allowing the structures to correctly interpret signals incoming from EGFR that are essential for eye and wing to acquire the final shape. No interaction was found with mutants involved in pathways other than the EGFR cascade (N, dpp and wg), making the role of ft in the control of the imaginal disc growth specifically dependent on its interaction with the EGFR pathway (Garoia, 2005).

The proximal ft clone allocation along the wing blade arises from an alteration in the growth direction and not from a different viability of the cells relative to their layout in the proximo-distal axis; it is then obvious that ft cells show a greater 'affinity' for the proximal region of the wing. The ft mutant phenotype seems anyway to be influenced by EGFR signalling also with respect to the proximalization of the clones; the distribution is indeed more homogeneous along the wing blade if the EGFR signalling is altered. It is interesting to notice that an EGFR signalling reduction or increase produces, in this case, the same biological effect, while the proliferative phenotype is directly correlated to the activity of the EGFR effectors. The activity of the EGFR cascade is spatially and temporally modulated during development, and in the wing disc it is gathered in the hinge and vein presumptive regions. Even if there are no evidences that the EGFR signalling plays a role in the P/D patterning of the wing blade, recent studies have shown that a gradient of EGFR activity is required for the correct P/D development of the leg. In the experimental conditions used, the MS1096-GAL4 driver creates an almost homogeneous EGFR signal along the wing blade, determining a quasi wild-type distribution of ft clones. This role in the modulation of the differential distribution of ft mutant cells along the P/D wing axis suggests for the EGFR pathway an involvement in the morphogenetic events that control the final shape of the wing (Garoia, 2005).

The ft-induced hyperplasia is associated with an abnormal pattern of gene expression, as visualized in two-dimensional protein gels of ft mutant imaginal discs. Although no data is available to hypothesize a cytoplasmic interaction between ft and the EGFR signalling, an alteration in MAPK expression could not be excluded. The ft mutant phenotypes, however, do not include the differentiation defects typical of the EGFR pathway genes, whose activity is indeed not altered since no significant modifications of the activated dpERK levels or patterns were detected in the ft tissues with respect to the wild-type (Garoia, 2005).

The results shown in this paper suggest that the interaction between ft and EGFR takes place at the proliferation level, while differentiation signals controlled by the EGFR pathway appear unaffected. With the aim to find some mechanisms that could explain the synergic phenotype of ft and EGFR mutations, the transcriptional levels of yan, dmyc and pnt, genes involved in proliferation control whose function is regulated by the EGFR cascade, were studied in ft and wild-type imaginal tissues. The results of semi-quantitative RT-PCR trials showed in ft tissues an increase of the transcription levels of yan and dmyc, whereas pnt was unaffected. The Dmyc transcription factor, the unique Drosophila homologue of the Myc family of proto-oncogenes, plays a central role in the control of cell growth in Drosophila. Overexpression of ras is capable to increase post-transcriptionally the Dmyc protein levels, promoting the G1-S transition via the increase of CycE translation. The increase in the Dmyc levels, however, affects growth rate but not proliferation, since the shortening of the G1 phase is balanced by the compensatory lengthening of G2, resulting in an increase in cell size but not in cell number. ft mutation otherwise induces an increase of cell proliferation without altering the cell size. Taken together, these results indicate that ft mutation affects not only the G1-S transition via Dmyc but also the G2-M transition, since the coordinated stimulation of the two cell-cycle checkpoints is necessary to increase the proliferation rate in Drosophila imaginal discs. Interestingly, the transcription level of pnt was unaffected in ft mutant discs. pnt is an ETS transcriptional activator that plays a central role in the mitosis control mediated by the EGFR signalling cascade; several studies however suggest the presence of additional Pnt-independent effectors in EGFR-mediated mitosis control. The ft control of the G2–M transition may involve EGFR effectors other than pnt, or molecules functioning through different signalling pathways. The yan gene is another component of the ETS transcriptional regulator family involved in the EGFR signalling. Phosphorylation by MAPK affects stability and subcellular localization of Yan, resulting in a rapid down-regulation of its activity. Yan functions as a fairly general inhibitor of differentiation, allowing both neuronal and non-neuronal cell types to choose between cell division and differentiation in multiple developmental contexts and recent studies indicate that the mammalian homologue of the Drosophila yan, TEL, is overexpressed in tumors. In the Drosophila developing eye yan is expressed in all undifferentiated cells and is down regulated as cells differentiate, so a high yan activity in ft mutant discs is correlatable with the observed proliferative advantage of ft cells (Garoia, 2005).

There are several indications that EGFR signalling can trigger different responses by different activity levels: in the Drosophila eye disc, differentiation requires high signalling levels, whereas lesser EGFR activity promotes mitosis and protects against cell death. These findings indicate that EGFR signalling may coordinate partially independent processes, transferring graded activity to the nucleus, rather than triggering 'all or none' responses. The simultaneous increase of activity in both growth promoters (dmyc) and differentiation repressors (yan) in ft mutant imaginal discs suggests the presence of a mechanism that shifts the EGFR nuclear equilibrium towards a level insufficient to induce differentiation but adequate for promoting cell growth and proliferation (Garoia, 2005).

These results indicate that, in the Drosophila imaginal discs, ft function is necessary for the correct interpretation of the multiple EGFR signals that coordinate proliferation, and that its loss causes misinterpretation of proliferation stimuli leading to tissue overgrowth. This effect may be due, at least in part, to the transcriptional regulation of genes involved in EGFR signalling. Nevertheless, the hyperplastic phenotype of ft mutations cannot be completely ascribed to its role in modulating signals transduced by EGFR, according to the very partial rescue observed utilizing dominant-negative alleles of the pathway. These results suggest that the ft function is not restricted to the modulation of EGFR signals, but controls different developmental events involved in imaginal discs morphogenesis (Garoia, 2005).

Several findings indicate that cadherin-catenin complexes may interact with growth factor receptors. The association of cadherins with growth factor receptors allows the assembly of a locally active apparatus that is essential for the generation of correct cell-cell signalling, as suggested by the downregulation of E-cadherins observed in mammalian tumors. Furthermore, E-cadherins were found to be a direct biochemical target of the EGFR pathway, suggesting a close relation of these molecules with the modulation of cell-cell communication. The only partial homology between the Ft protocadherin and the classic E-cadherins, and the lack of data about interactors for the cytoplasmic domain of ft makes a direct comparison of their function very difficult. Taken together, the data suggest a novel mechanism through which ft tumor suppressor gene and EGFR pathway cooperate in the control of proliferation and morphogenesis in Drosophila imaginal tissues (Garoia, 2005).

The orientation of cell divisions determines the shape of Drosophila organs: Mutations in the Drosophila PCP genes dachsous (ds) and fat (ft) cause changes in the shape of different organs

Organ shape depends on the coordination between cell proliferation and the spatial arrangement of cells during development. Much is known about the mechanisms that regulate cell proliferation, but the processes by which the cells are distributed in an orderly manner remain unknown. This can be accomplished either by random division of cells that later migrate locally to new positions (cell allocation) or through polarized cell division (oriented cell division; OCD). Recent data suggest that the OCD is involved in some morphogenetic processes such as vertebrate gastrulation, neural tube closure, and growth of shoot apex in plants; however, little is known about the contribution of OCD during organogenesis. The orientation patterns of cell division was examined throughout the development of wild-type and mutant imaginal discs of Drosophila. The results show a causal relationship between the orientation of cell divisions in the imaginal disc and the adult morphology of the corresponding organs, indicating a key role for OCD in organ-shape definition. In addition, a subset of planar cell polarity genes was found to be required for the proper orientation of cell division during organ development (Baena-López, 2005).

Drosophila imaginal discs are a classical model system for studying general mechanisms involved in the control of organ growth and patterning. The imaginal discs are epithelial structures that originate from the embryonic ectoderm, and, after a period of cell proliferation during the larval stages, give rise to most adult organs. The wing disc is divided into lineage units known as compartments. The boundaries between compartments play a key role in the control of wing disc growth and patterning. Analysis of mitotic recombination clones in animals and plants allows for tracing the descendants of single marked cells during development. These experiments have shown a clear correlation between the shape of the clones and adult morphology of the organ where the clones are studied, i.e., clone growth defines organ shape. Most clones in the wing blade are very elongated and grow along the proximal-distal (P/D) axis of the wing, perpendicular to the D/V border. In contrast, clones within the wing margin grow along the D/V border. This study has considered the dorsal-ventral (D/V) boundary as a reference to measure the orientation of cell divisions during wing disc development. A striking relationship was observed between the shape of the clones and the orientation of cell divisions. Thus, the majority of cells divide along the proximal-distal (P/D) axis of the wing blade; 59.4% (n = 549) of mitoses form angles higher than 55° with respect to the D/V boundary, while only 13.8% are lower than 35°. In contrast, in the wing margin, most cells divide nearly parallel to the D/V boundary, forming angles lower than 35° (71.4%; n = 70). Furthermore, the characteristic shape of each intervein region is also reflected in the cell-orientation patterns. Thus, 65.8% (n = 116) of the mitotic figures studied in intervein regions C and D, where clones are very elongated and grow perpendicular to the wing margin, are nearly perpendicular to the D/V border, whereas in regions A and E, where clones are wider and grow parallel to the D/V border, only 50.3% of mitoses (n = 81) have this orientation (Baena-López, 2005).

Early-induced clones also show an elongated shape along the P/D axis of the wing. Accordingly, most of the cell divisions in the second instar wing discs appear with a P/D orientation. In everted wings in pupae, most cells also divide preferentially along the P/D axis, indicating that the correlation between the orientation of cell divisions and the shape of the clones is maintained throughout development (Baena-López, 2005).

Interestingly, the orientation of postmitotic daughter cells, analyzed in clones of two cells, conserves the positions determined by the angle of the OCD. The orientation of the first cell division tends to be maintained in subsequent divisions, since it is observed that 57% (n = 121) of clones of four cells form straight lines of one cell width. Although these results suggest that the cell relocation plays a minor role to define the clone shape, it cannot be ruled out that this process might refine clone shapes and therefore organ shape. Finally, it is suggested that the width of the clones mainly depends on the general probability of OCD (Baena-López, 2005).

To evaluate the general requirement of OCD in organogenesis, the patterns of mitotic orientation were examined in other wing disc regions. The thorax shows an isodiametric morphology, and mitotic recombination clones grow isodiametrically. Accordingly, no preferential orientation of the planar axes of cell divisions was found. In the peripodial epithelium, it was also observed that the orientation of cell divisions define the characteristic shape of the clones. As in the wing blade, postmitotic cells show the same orientation of previous cell divisions (Baena-López, 2005).

The general requirement of the OCD in the definition of wing disc morphology led to a study of the contribution of this process to the development of other organs. The pattern of cell-division orientations was examined during eye disc development. The dorsoventral midline of the Drosophila eye is known as the equator and defines a line of mirror-image symmetry, with ommatidia on each side having opposite chirality. This clonal boundary also plays an important role in the patterning and growth of the eye. Clones in the eye grow symmetrically oblique with respect to the equator. During eye development, an indentation known as the morphogenetic furrow (MF) marks the front of a wave of differentiation that sweeps from posterior to anterior across the disc. The orientation of cell divisions was monitored with respect to the MF in the region anterior to the furrow. The orientation of mitotic spindles in most mitotic figures in the ventral half of the eye discs form angles between 40° and 60°, whereas in the dorsal half, they form angles between 120° and 140°. In the eye disc, the postmitotic cell allocation again reflects the orientation of mitosis. Attempts were made to measure the orientation of cell divisions in a tube-shaped organ like the leg, but the highly folded epithelial organization of these discs prevented the reaching of any clear conclusion. However, clones in the adult legs appear narrow, running proximodistally over several joints. The results indicate that the OCD may be a general mechanism to generate shape during organogenesis (Baena-López, 2005).

One interesting group of genes involved in the orientation of cell divisions during Zebrafish gastrulation and growth of shoot apex in Arabidopsis is composed of planar cell polarity (PCP) genes. The function of these genes is evolutionary conserved to define the polarity and positional information of the cells within an epithelium. Mutations in the Drosophila PCP genes dachsous (ds) and fat (ft) cause changes in the shape of different organs: wings and eyes are rounder than the wild-type, whereas legs are wider and shorter. The activity of these genes is required upstream of other PCP genes that do not affect adult organ shape such as the core PCP genes strabismus (stbm), prickled (pk), and flamingo (fmi) or the effector PCP gene multiple wing hair (mwh). Whether the abnormal organ shapes, observed in ds and ft mutant backgrounds, are associated with changes in the clone shape and the orientation of cell division was analyzed (Baena-López, 2005).

Mitotic recombination clones in ds mutant wing discs show rounded shapes, losing the elongated shape of wild-type clones. Interestingly, loss-of-function clones for ds or ft fail to adopt their typical enlarged shape in the wing blade or in the eye, showing a rounder shape than wild-type clones. In contrast to control wing discs and wild-type clones, it was observed that the orientation of cell divisions is randomized in ds mutant cells (wing discs or ds mutant clones). Accordingly, postmitotic cells do not show any preferential orientation. The rounded shape and disturbed OCD is also observed in clones of ds- or ft-expressing cells and in adult wings where ds or ft is ectopically expressed using the UAS/G4 system. Similar results are observed in the eye imaginal discs. These results suggest that the polarization cues mediated by PCP genes are required for the control of the orientation of cell division and, therefore, are involved in organ shape definition. These findings show that some of the genes required for PCP are acting in early stages of the development, suggesting that the base for PCP may be established during larval development. The conserved function of PCP genes controlling OCD in different organisms suggests that this process might be an evolutionary mechanism in organ shape definition. Although the mechanistic details underlying the new functions of PCP genes are unknown, it is likely that the asymmetric cell distribution of a classical core of PCP proteins is not required. Finally, similar phenotypes were observed in both the lack and excess of function for ds and ft, suggesting that the default organ shape is circular when the cell polarization is disrupted (Baena-López, 2005).

To conclude, it is proposed that the shape of organs can be accounted for by the oriented pattern of cell divisions rather than postmitotic relocation of proliferating cells. However, how the preferential orientation is topologically determined is poorly understood. It is speculated that the orientation of cell divisions in different territories during development may result from the integration of signals coming from restriction borders and territorial local cell interactions. The existence of clusters of cells, synchronized in the same stage of the cell cycle, support the idea of a local control of cell proliferation. It has been found that the orientations of cell divisions in these clusters tend to be highly aligned, suggesting also a local control of OCD superimposed to more general and long-range signals. Although the signals exchanged between neighboring cells to determine local OCD are still unknown, it is likely that a subset of PCP genes could be involved in this process (Baena-López, 2005).

Separating the adhesive and signaling functions of the Fat and Dachsous protocadherins

The protocadherins Fat (Ft) and Dachsous (Ds) are required for several processes in the development of Drosophila, including controlling growth of imaginal discs, planar cell polarity (PCP) and the proximodistal patterning of appendages. Ft and Ds bind in a preferentially heterophilic fashion, and Ds is expressed in distinct patterns along the axes of polarity. It has thus been suggested that Ft and Ds serve not as adhesion molecules, but as receptor and ligand in a poorly understood signaling pathway. To test this hypothesis, a structure-function analysis of Ft and Ds was performed, separating their adhesive and signaling functions. It was found that the extracellular domain of Ft is not required for its activity in growth, PCP and proximodistal patterning. Thus, ligand binding is not necessary for Ft activity. By contrast, the extracellular domain of Ds is necessary and sufficient to mediate its effects on PCP, consistent with the model that Ds acts as a ligand during PCP. However, evidence is also provided that Ds can regulate growth independently of Ft, and that the intracellular domain of Ds can affect proximodistal patterning, both suggestive of functions independent of binding Ft. Finally, it is shown that ft mutants or a dominant-negative Ft construct can affect disc growth without changes in the expression of wingless and Wingless target genes (Matakatsu, 2006).

Chief amongst the findings of this study is that Ft activity is not simply a byproduct of changes in cell-cell adhesion. The FtΔECD construct lacks almost the entire extracellular domain and cannot bind or stabilize Ds in vitro or in vivo. Nonetheless, it can rescue the lethality, overgrowth and PCP defects of ft alleles that should be null for any adhesive or receptor function, and in a wild-type background can disrupt proximodistal patterning. This suggests that the intracellular domain of Ft can act in the absence of binding between endogenous Ft and Ds, or indeed between Ft and any other extracellular ligand, as long as sufficient levels are expressed (Matakatsu, 2006).

Conversely, it was found that a form of Ft lacking the intracellular domain (FtΔICD) failed to rescue overgrowth in ft mutants. In fact, this form acts as a strong dominant negative, inducing overgrowth of wild-type and ft mutant imaginal discs. This occurs despite the ability of FtΔICD to stabilize endogenous cell surface Ds and Ft, raising the possibility that FtΔICD binds to Ds and Ft is a way that blocks their activities. The possibility that FtΔICD alters the activity of some additional, unknown player cannot be ruled out. Although lethality prevents determining whether FtΔICD can rescue ft mutant PCP defects, expression of FtΔICD in wild-type wings also disrupts PCP. These PCP defects are weaker than those observed in ft mutants, suggesting that FtΔICD might have stronger effects on growth control than PCP (Matakatsu, 2006).

In contrast to Ft, the extracellular domain of Ds is sufficient for its effects on PCP. The DsΔICD construct lacks almost the entire intracellular domain, but nonetheless can rescue the PCP defects of strong ds mutants and disrupt PCP in wild-type wings. The DsΔECD construct, however, cannot bind or stabilize Ft and cannot rescue ds mutant PCP defects or influence PCP in wild-type wings. The results thus support the hypothesis that in PCP Ds acts chiefly as a ligand for Ft, modulating its activity (Matakatsu, 2006).

Nonetheless, the possibility that the intracellular domain of Ds has some PCP activity within the context of the whole protein cannot be ruled out, and the conservation of large regions of the Ds intracellular domain in its vertebrate homologs dachsous 1 and dachsous 2 suggests that Ds may have activity beyond that of a ligand. Thus, it is intriguing that expression of DsΔECD can disrupt another ds-sensitive phenotype, crossvein spacing in wild-type wings. Since crossvein spacing defects can result from either gains or losses in Ds or Ft function, it is possible that this defect is caused by disrupting the function of endogenous Ds, and thus the ability of that Ds to signal via Ft. However, DsΔECD did not cause any obvious change in the levels of endogenous Ds. Moreover, loss of Ds normally causes visible destabilization of cell surface Ft, and no changes were seen in Ft levels in cells misexpressing DsΔECD (Matakatsu, 2006). ds mutations can also enhance the overgrowth observed in mutants that lack the intracellular domain of Ft, indicating that in overgrowth, Ds activity is not completely dependent on regulating the activity of the intracellular domain of Ft. In this respect, overgrowth differs from PCP; ft mutants and ds ft double mutants produce identical PCP phenotypes. The result could be explained if Ds regulates growth via its intracellular domain. Alternatively, Ds may be acting as an extracellular ligand for a binding partner other than Ft (Matakatsu, 2006).

The results support the hypothesis the Ft signals via its intracellular domain in growth control, PCP and proximodistal patterning. Similarly, it is likely that the intracellular domain of Ds contributes to proximodistal patterning and perhaps growth control. The conservation of long stretches of the intracellular domain of Ft and Ds in the vertebrate homologs Fat4, dachsous 1 and dachsous 2 also suggests that there is conserved binding to intracellular factors (Matakatsu, 2006).

There are no known binding partners for the intracellular domain of Ds or dachsous-like proteins. The intracellular domain of Drosophila Ft also lacks the ENA-VASP binding sites that mediate at least some of the function of vertebrate Fat1 in vitro. The intracellular domain of Drosophila Ft can bind the atrophin Grunge, and genetic evidence suggests a link between Grunge and PCP. However, it is not yet clear if Grunge acts downstream of Ft, nor is it clear how atrophins, which act as transcriptional co-repressors, could polarize cells. grunge mutants also do not apparently reproduce the effects of ft mutants on disc growth or on wg expression in the prospective wing hinge (Matakatsu, 2006).

Some evidence suggests that Ds and Ft regulate growth and patterning by altering either the expression of wg in the prospective wing hinge or the response to Wg signaling. However, the current results make it unlikely that this can explain all but a small part of the overgrowth phenotype. The overgrowth induced by ft mutations or FtΔICD occurs without any consistent change in the expression of Wg target genes Dll or Vg, or in the expression of wg. Moreover, FtΔICD induced overgrowth in the entire wing disc, but whereas increased Wg signaling can induce overgrowth in the hinge, in the prospective wing blade Wg signaling reduces growth. The results are consistent with the failure of mutants in the Wg signaling pathway to modify the ft overgrowth phenotype (Matakatsu, 2006).

A recent study has suggested a possible link between overgrowth and Ras signaling; mild reductions in Ras function that have little effect on the growth of wild-type cells can block the overgrowth observed in ft mutant clones. It remains to be seen whether Ft can actually affect Ras signaling, or whether this represents the convergence of the two pathways on a shared target (Matakatsu, 2006).

Because Ds is expressed in an apparently graded fashion along the axes of polarity, it was suggested that Ds provides a global cue that orients PCP in the eye, wing and abdomen. But whereas patterned Ds misexpression is sufficient to reorient PCP, and patterned Ds expression does appear to be necessary for normal PCP in the eye, in the wing uniform Ds expression is able to rescue most of the ds mutant PCP defects. This suggests that most of the PCP defects in ds mutant wings are caused, not by a change in the spatial regulation of Ds-Ft signaling, but rather by the loss of a basal level of signaling required for the proper activity of some other polarizing cue. These results left open the possibility that Ft activity is being spatially regulated by an extracellular ligand other than Ds. However, this study shows that ft mutant PCP defects can be substantially rescued by uniform expression of FtΔECD, a form of Ft that cannot bind Ds, or probably any other ligand (Matakatsu, 2006).

There is, however, a region in the proximal wing where PCP defects cannot be rescued with uniform expression of either Ds, Ft, or FtΔECD. This is also the region of the wing where there is a boundary or sharp gradient between proximal regions with high and distal regions with low ds expression. Thus, it remains possible that Ds and Ft activities are permissive in much of the wing but, in the proximal wing, spatially instructive. The different sensitivities of different regions to changes in Ds and Ft may reflect localized differences in the strength of other partially redundant polarizing cues (Matakatsu, 2006).

Regulation of leg size and shape by the Dachsous/Fat signalling pathway during regeneration

An amputated cricket leg regenerates all missing parts with normal size and shape, indicating that regenerating blastemal cells are aware of both their position and the normal size of the leg. However, the molecular mechanisms regulating this process remain elusive. This study used a cricket model (the two-spotted cricket, Gryllus bimaculatus) to show that the Dachsous/Fat (Ds/Ft) signalling pathway is essential for leg regeneration. Knockdown of ft or ds transcripts by regeneration-dependent RNA interference (rdRNAi) suppressed proliferation of the regenerating cells along the proximodistal (PD) axis concomitantly with remodelling of the pre-existing stump, making the regenerated legs shorter than normal. By contrast, knockdown of the expanded (ex) or Merlin (Mer) transcripts induced over-proliferation of the regenerating cells, making the regenerated legs longer. These results are consistent with those obtained using rdRNAi during intercalary regeneration induced by leg transplantation. A model is presented to explain these results in which the steepness of the Ds/Ft gradient controls growth along the PD axis of the regenerating leg (Bando, 2009).

Regeneration-dependent RNAi is a type of RNAi that occurs specifically after leg amputation in cricket nymphs that have been injected with double-stranded RNA (dsRNA) for a target gene (Nakamura, 2008a). In this system, when the metathoracic (T3) tibia of the third-instar nymph is amputated, it takes approximately 40 days (six ecdyses) to restore the adult leg. The process begins with the covering of the amputated region by newly formed cuticle. A ligand of Epidermal growth factor receptor (Gb'Egfr) is then induced by Decapentaplegic (Gb'Dpp) and Wingless (Gb'Wg) in a blastema composed of epithelial stem cells, which begins to undergo rapid proliferation to restore the lost portion in the fourth instar (Mito, 2002; Nakamura, 2008b). In the fifth instar, the tibiae, tibial spurs, tarsi and tarsal claws are restored in miniature. In the seventh instar, the amputated legs restore the missing portion to regain a nearly normal appearance. As no leg regeneration was observed after amputation in the case of rdRNAi against Gb'armadillo (Gb'arm), the canonical Wnt pathway should be involved in the initiation of the regeneration (Nakamura, 2007; Bando, 2009 and references therein).

Using an RNAi knockdown approach against 23 candidate genes, this study identified 15 components of the Ds/Ft signalling pathway that are involved in cricket leg regeneration. Based on additional data from Gryllus and Drosophila, a model signalling cascade was proposed for the regulation of leg regeneration by the Ds/Ft signalling pathway. As the main components of the Ds/Ft signalling pathways are conserved in vertebrates, this signalling cascade may also be involved in vertebrate leg regeneration (Bando, 2009).

The most typical phenotypes were the short and thick legs induced by rdRNAi against Gb'ft or Gb'ds. It is known that the size of each leg segment normally scales with overall body size, a phenomenon known as allometry. Surprisingly, the size of the regenerated legs in the phenotypes that were observed did not scale with overall body size. Furthermore, the size of the regenerated legs depended upon the site of tibial amputation. It is noteworthy that, although the expression patterns of Gb'ft and Gb'ds were different, their short and thick phenotypes were similar. This is consistent with the fact that Drosophila mutant phenotypes of both ft and ds in adult legs are short and thick, despite the fact that ft and ds have distinct expression patterns in Drosophila imaginal discs. Thus, it is concluded that the activity of Ds/Ft signalling regulates leg segment size and shape during regeneration. Furthermore, it was shown that the Ds/Ft signalling pathway may regulate leg size during regeneration through the Hpo signalling pathway. This is also supported by the fact that the Hpo signalling pathway is involved in an intrinsic mechanism that restricts organ size and that the Ds/Ft signalling system defines a cell-to-cell signalling mechanism that regulates the Hpo pathway, thereby contributing to the control of organ size (Bando, 2009).

Meinhardt pointed out that two processes operate during leg regeneration. One, which operates during the restoration of distal structures, is instructed by a morphogen epidermal growth factor (Egf), which is itself induced by two morphogens, Dpp and Wg, at the amputated surface (Meinhardt, 1982; Mito, 2002; Nakamura, 2008a). The other, operating in intercalary regeneration, is directly controlled by neighbouring cells at the junction between host and graft, but not by long-range morphogens (Meinhardt, 2007; Nakamura, 2008a). It is likely that the Ds/Ft signalling pathway participates in both mechanisms, because rdRNAi against Gb'ft or Gb'ds affected leg regeneration after either distal amputation or intercalary transplantation. In the case of distal amputation, as the Gryllus tarsi and claws were not restored after tibial amputation in the Gb'Egfr rdRNAi nymphs (Nakamura, 2008a), it has been speculated that Gb'Egf functions as a morphogen in the leg regeneration, as found in Drosophila leg imaginal discs. Recently, it has been demonstrated in the Drosophila wing disc that the Fat signalling pathway links the morphogen-mediated establishment of gradients of positional values across developing organs to the regulation of organ growth. Thus, it is speculated that the Ds/Ft system links the Egf-mediated establishment of gradients of positional values across regenerating blastemal cells to the regulation of regenerate growth (Bando, 2009).

As Gb'd rdRNAi legs exhibited the short-leg phenotypes, but not thick ones, and Gb'd (decapentaplegic) is epistatic of Gb'ft and Gb'ds, Gb'D may mediate the components of Ds/Ft signalling controlling leg size. The enlarged phenotype of Gb'wts RNAi nymphs was suppressed by RNAi against Gb'd in Gryllus, indicating that Gb'd is downstream of Gb'wts. This result differs from Drosophila data. A genetic analysis is necessary to confirm the difference, because the epistatic allele is not null in RNAi experiments. As the phenotype of rdRNAi treatment against Gb'ds was weaker than that against Gb'ft, as reported in the corresponding Drosophila mutants, Gb'Ft may interact with factors that are as yet unidentified. Although the effect of rdRNAi against Gb'fj on leg size was very mild, the possible involvement of Gb'fj in allometric growth cannot be excluded. The short phenotypes were observed in the Gb'sd RNAi nymphal legs, so it remains a possibility that Gb'sd is involved in allometric leg growth. However, the apparent contribution of the Hpo-Sav-Mats complex is as yet uncertain (Bando, 2009).

Regenerated legs of Gb'ex and Gb'Mer RNAi adults become longer than normal control legs, and Gb'ex and Gb'Mer regulate cell proliferation induced by the presence of positional disparity. These results suggest that Gb'ex and Gb'Mer are also involved in allometric growth of the leg segment. In Drosophila, Ex and Mer negatively regulate cell growth and proliferation through the Hpo/Wts pathway. In mammalian cells, Nf2 (merlin) is known to be a crucial regulator of contact-dependent inhibition of proliferation (Curto, 2008). Thus, it is concluded that activities of Ex and Mer may regulate contact-dependent inhibition of proliferation via the Wts signalling pathway to restore the proper leg segment size during regeneration (Bando, 2009).

A widely accepted model for leg regeneration is the intercalation model, based on positional information. This model is based on the intercalation of new structures so as to re-establish continuity of positional values during regeneration. However, on the basis of this model, it is difficult to explain the changes in leg size that were observed in the present study. Thus, the model need to be extended to include the control of growth and tissue size during regeneration. Several models have been proposed to explain how organ size is regulated. Lawrence (2008) proposed a model, which is referred to here as the Ds/Ft steepness model, to explain the mechanisms underlying the determination of organ size and PCP, including the Warts/Hippo pathway as the mechanism for controlling growth. In this model, it was hypothesized that: (1) the morphogens responsible for the overall pattern of an organ establish and orient the Ds/Ft system, which then forms a linear Ds/Ft gradient. The nature of the Ds/Ft gradient is unknown, although the number of Ds/Ft trans heterodimers is the key variable; (2) the steepness of the Ds/Ft gradient regulates Hpo target expression and cell proliferation, and its direction provides information used to establish the correct cellular polarity; (3) growth would be expected to cease when the slope of the gradient declines below a certain threshold value; and (4) the maximum and minimum limits of the system are conserved, while recently divided cells take up intermediate scalar values from their neighbours (Bando, 2009).

Using this model, a modified Ds/Ft steepness model is proposed for leg regeneration acting as follows. The results indicate that nymphal leg regeneration depends on two major processes: (1) proliferation and differentiation of blastemal cells and (2) growth of the pre-existing stump. In each of these processes, new positional identities are specified in relation to new segment boundaries. According to the Ds/Ft steepness model, in normal regeneration, a very steep gradient should be formed in the regenerating blastema. The regenerate may grow so as to restore the normal pre-existing steepness. Reassignment of positional identities after amputation will correlate with a similar re-setting of the minimum Ds/Ft scalar value, and the results are consistent with the steepness hypothesis (Bando, 2009).

Growth of the pre-existing stump is a normal component of leg growth, in which the pre-existing stump cells proliferate according to some allometric signals, which may be related to the maximum scalar value and a slope of the gradient, keeping their original positional information. This was observed in the truncated leg of Gb'arm rdRNAi adults (Nakamura, 2007; Bando, 2009).

In the absence of the proliferation and differentiation of blastemal cells, as observed in the Gb'ft rdRNAi leg, the minimum scalar value, which is the most distal positional value, would be established at the site of amputation, and the Ds/Ft gradient would be expected, in turn, to shift down with the same slope as the pre-existing one. The Ds/Ft steepness model provides an explanation for the observation that the final leg size depends on the amputated position, if it is assumed that the gradient shifts down with the same slope as that where cells at an amputated position have the minimum scalar value. Thus, the observed re-specification of regeneration legs induced in the legs treated with rdRNAi against Gb'ft or Gb'ds is as would be predicted by the Ds/Ft steepness model. Thus, it is likely that the Ds/Ft gradient functions to link positional and allometric information to the regulation of leg segment growth. Furthermore, if it is assumed that the activity of Ex/Mer is related to a threshold value of the slope of the gradient that determines when growth ceases, all rdRNAi phenotypes in the present study can be interpreted consistently with the Ds/Ft steepness model for regeneration (Bando, 2009).

A feed-forward circuit linking wingless, fat-dachsous signaling, and the warts-hippo pathway to Drosophila wing growth

During development, the Drosophila wing primordium undergoes a dramatic increase in cell number and mass under the control of the long-range morphogens Wingless (Wg, a Wnt) and Decapentaplegic (Dpp, a BMP). This process depends in part on the capacity of wing cells to recruit neighboring, non-wing cells into the wing primordium. Wing cells are defined by activity of the selector gene vestigial (vg) and recruitment entails the production of a vg-dependent 'feed-forward signal' that acts together with morphogen to induce vg expression in neighboring non-wing cells. This study identifies the protocadherins Fat (Ft) and Dachsous (Ds), the Warts-Hippo tumor suppressor pathway, and the transcriptional co-activator Yorkie (Yki, a YES associated protein, or YAP) as components of the feed-forward signaling mechanism; this mechanism promotes wing growth in response to Wg. vg generates the feed-forward signal by creating a steep differential in Ft-Ds signaling between wing and non-wing cells. This differential down-regulates Warts-Hippo pathway activity in non-wing cells, leading to a burst of Yki activity and the induction of vg in response to Wg. It is posited that Wg propels wing growth at least in part by fueling a wave front of Ft-Ds signaling that propagates vg expression from one cell to the next (Zecca, 2010).

During larval life, the Drosophila wing primordium undergoes a dramatic ~200-fold increase in cell number and mass driven by the morphogens Wg and Dpp. Focusing on Wg, it has been established that this increase depends at least in part on a reiterative process of recruitment in which wing cells send a feed-forward (FF) signal that induces neighboring cells to join the primordium in response to morphogen. The present results identify Ft-Ds signaling, the Wts-Hpo tumor suppressor pathway, and the transcriptional co-activator Yki as essential components of the FF process and define the circuitry by which it propagates from one cell to the next. This discussion considers, in turn, the nature of the circuit, the parallels between FF signaling and PCP, and the implications for the control of organ growth by morphogen (Zecca, 2010).

Several lines of evidence are presented that expression of the wing selector gene vg drives production of the FF signal by promoting a non-autonomous signaling activity of Ft. First, it was shown that vg acts both to up-regulate fj and down-regulate ds, two outputs known to elevate an outgoing, signaling activity of Ft in PCP. Second, it was demonstrated that experimental manipulations that elevate Ft signaling -- specifically, over-expression of Ft or removal of Ds -- generate ectopic FF signal. Third, and most incisively, it was shown that ft is normally essential in wing cells to send FF signal (Zecca, 2010).

Ft and Ds are both required in non-wing cells to receive the FF signal, functioning in this capacity to prevent the activation of vg unless countermanded by FF input. Notably, the removal of either Ft or Ds from non-wing cells constitutively activates the FF signal transduction pathway, mimicking receipt of the FF signal. However, the pathway is only weakly activated in this condition and the cells are refractory to any further elevation in pathway activity (Zecca, 2010).

Previous studies have defined a transduction pathway that links Ft-Ds signaling via the atypical myosin myosin Dachs (D) to suppression of the Wts kinase and enhanced nuclear import of Yki. Likewise, Ft and Ds operate through the same pathway to transduce the FF signal. Specifically, it was shown that manipulations of the pathway that increase nuclear activity of Yki (over-expression of D or Yki, or loss of Wts or Ex) cause non-wing cells to adopt the wing state. Conversely, removal of D, an intervention that precludes down-regulation of Wts by Ft-Ds signaling, prevents non-wing cells from being recruited into the wing primordium (Zecca, 2010).

To induce non-wing cells to become wing cells, transduction of the FF signal has to activate vg transcription. Activation is mediated by the vg QE and depends on binding sites for Scalloped (Sd), a member of the TEAD/TEF family of DNA binding proteins that can combine with either Yki or Vg to form a transcriptional activator Hence, it is posited that Yki transduces the FF signal by entering the nucleus and combining with Sd to activate vg. In addition, it is posited that once sufficient Vg produced under Yki-Sd control accumulates, it can substitute for Yki to generate a stable auto-regulatory loop in which Vg, operating in complex with Sd, sustains its own expression. Accordingly, recruitment is viewed as a ratchet mechanism. Once the auto-regulatory loop is established, neither FF signaling nor the resulting elevation in Yki activity would be required to sustain vg expression and maintain the wing state (Zecca, 2010).

Both the activation of the QE by Yki as well as the maintenance of its activity by Vg depend on Wg and Dpp input and hence define distinct circuits of vg auto-regulation fueled by morphogen. For activation, the circuit is inter-cellular, depending on Ft-Ds signaling for vg activity to propagate from one cell to the next. For maintenance, the circuit is intra-cellular, depending on Vg to sustain its own expression. Accordingly, it is posited that growth of the wing primordium is propelled by the progressive expansion in the range of morphogen, which acts both to recruit and to retain cells in the primordium (Zecca, 2010).

To date, Ft-Ds signaling has been studied in two contexts: the control of Yki target genes in tissue growth and the orientation of cell structures in PCP. Most work on tissue growth has focused on Yki target genes that control basic cell parameters, such as survival, mass increase, and proliferation (e.g., diap, bantam, and cyclinE). In this context, Ds and Ft are thought to function as a ligand-receptor pair, with tissue-wide gradients of Ds signal serving to activate Ft to appropriate levels within each cell. In contrast, Ft and Ds behave as dual ligands and receptors in PCP, each protein having intrinsic and opposite signaling activity and both proteins being required to receive and orient cells in response to each signal (Zecca, 2010).

This study has analyzed a different, Yki-dependent aspect of growth, namely the control of organ size by the regulation of a selector gene, vg. In this case, Ft appears to correspond to a ligand, the FF signal, and Ds to a receptor required to receive the ligand -- the opposite of the Ds-Ft ligand-receptor relationship inferred to regulate other Yki target genes. Moreover, as in PCP, evidence was found that Ft and Ds operate as bidirectional ligands and receptors: like Ds, Ft is also required for receipt of the FF signal, possibly in response to an opposing signal conferred by Ds (Zecca, 2010).

Studies of Ft-Ds interactions, both in vivo and in cell culture, have established that Ft and Ds interact in trans to form hetero-dimeric bridges between neighboring cells, the ratio of Ft to Ds presented on the surface of any given cell influencing the engagement of Ds and Ft on the abutting surfaces of its neighbors. These interactions are thought, in turn, to polarize the sub-cellular accumulation and activity of D. Accordingly, it is posited that vg activity generates the FF signal by driving steep and opposing differentials of Ft and Ds signaling activity between wing (vg^ON) and non-wing (vg^OFF) cells. Further, it is posited that these differentials are transduced in cells undergoing recruitment by the resulting polarization of D activity, acting through the Wts-Hpo pathway and Yki to activate vg (Zecca, 2010).

Thus, it is proposeed that FF propagation and PCP depend on a common mechanism in which opposing Ft and Ds signals polarize D activity, both proteins acting as dual ligands and receptors for each other. However, the two processes differ in the downstream consequences of D polarization. For FF propagation, the degree of polarization governs a transcriptional response, via regulation of the Wts-Hpo pathway and Yki. For PCP, the direction of polarization controls an asymmetry in cell behavior, through a presently unknown molecular pathway (Zecca, 2010).

FF propagation and PCP may also differ in their threshold responses to D polarization. vg expression is graded, albeit weakly, within the wing primordium, due to the response of the QE to graded Wg and Dpp inputs. Hence, a shallow differential of Ft-Ds signaling reflecting that of Vg may be sufficient to orient cells in most of the prospective wing territories, but only cells in the vicinity of the recruitment interface may experience a steep enough differential to induce Yki to enter the nucleus and activate vg (Zecca, 2010).

Finally, FF propagation and PCP differ in at least one other respect, namely, that they exhibit different dependent relationships between Ft and Ds signaling. In PCP, clonal removal of either Ft or Ds generates ectopic polarizing activity, apparently by creating an abrupt disparity in the balance of Ft-to-Ds signaling activity presented by mutant cells relative to that of their wild type neighbors. By contrast, in FF propagation, only the removal of Ds, and not that of Ft, generates ectopic FF signal. This difference is attributed to the underlying dependence of Ft and Ds signaling activity on vg. In ds^o cells, Ft signaling activity is promoted both by the absence of Ds and by the Vg-dependent up-regulation of fj. However, in ft^o cells, Ft is absent and Vg down-regulates ds, rendering the cells equivalent to ds^o ft^o cells (which are devoid of signaling activity in PCP). Thus, for FF propagation, the underlying circuitry creates a context in which only the loss of Ds, but not that of Ft, generates a strong, ectopic signal. For PCP, no such circuit bias applies (Zecca, 2010).

Morphogens organize gene expression and cell pattern by dictating distinct transcriptional responses at different threshold concentrations, a process that is understood conceptually, if not in molecular detail. At the same time, they also govern the rate at which developing tissues gain mass and proliferate, a process that continues to defy explanation (Zecca, 2010).

One long-standing proposal, the 'steepness hypothesis,' is that the slope of a morphogen gradient can be perceived locally as a difference in morphogen concentration across the diameter of each cell, providing a scalar value that dictates the rate of growth. Indeed, in the context of the Drosophila wing, it has been proposed that the Dpp gradient directs opposing, tissue-wide gradients of fj and ds transcription, with the local differential of Ft-Ds signaling across every cell acting via D, the Wts-Hpo pathway, and Yki to control the rate of cell growth and proliferation. The steepness hypothesis has been challenged, however, by experiments in which uniform distributions of morphogen, or uniform activation of their receptor systems, appear to cause extra, rather than reduced, organ growth (Zecca, 2010).

The current results provide an alternative interpretation (see The vestigial feed-forward circuit, and the control of wing growth by morphogen). It is posited that 'steepness,' as conferred by the local differential of Ft-Ds signaling across each cell, is not a direct reflection of morphogen slope but rather an indirect response governed by vg activity. Moreover, it is proposed that it promotes wing growth not by functioning as a relatively constant parameter to set a given level of Wts-Hpo pathway activity in all cells but rather by acting as a local, inductive cue to suppress Wts-Hpo pathway activity and recruit non-wing cells into the wing primordium (Zecca, 2010).

How important is such local Ft-Ds signaling and FF propagation to the control of wing growth by morphogen? In the absence of D, cells are severely compromised for the capacity to transduce the FF signal, and the wing primordium gives rise to an adult appendage that is around a third the normal size, albeit normally patterned and proportioned. A similar reduction in size is also observed when QE-dependent vg expression is obviated by other means. Both findings indicate that FF signaling makes a significant contribution to the expansion of the wing primordium driven by Wg and Dpp. Nevertheless, wings formed in the absence of D are still larger than wings formed when either Wg or Dpp signaling is compromised. Hence, both morphogens must operate through additional mechanisms to promote wing growth (Zecca, 2010).

At least three other outputs of signaling by Wg (and likely Dpp) have been identified that work in conjunction with FF propagation. First, as discussed above, Wg is required to maintain vg expression in wing cells once they are recruited by FF signaling, and hence to retain them within the wing primordium. Second, it functions to provide a tonic signal necessary for wing cells to survive, gain mass, and proliferate at a characteristic rate. And third, it acts indirectly, via the capacity of wing cells, to stimulate the growth and proliferation of neighboring non-wing cells, the source population from which new wing cells will be recruited. All of these outputs, as well as FF propagation, depend on, and are fueled by, the outward spread of Wg and Dpp from D-V and A-P border cells. Accordingly, it is thought that wing growth is governed by the progressive expansion in the range of Wg and Dpp signaling (Zecca, 2010).

Identification of Ft-Ds signaling, the Wts-Hpo pathway, and Yki as key components of the FF recruitment process provides a striking parallel with the recently discovered involvement of the Wts-Hpo pathway and Yki/YAP in regulating primordial cell populations in vertebrates, notably the segregation of trophectoderm and inner cell mass in early mammalian embryos and that of neural and endodermal progenitor cells into spinal cord neurons and gut. As in the Drosophila wing, Wts-Hpo activity and YAP appear to function in these contexts in a manner that is distinct from their generic roles in the regulation of cell survival, growth, and proliferation, namely as part of an intercellular signaling mechanism that specifies cell type. It is suggested that this novel employment of the pathway constitutes a new, and potentially general, mechanism for regulating tissue and organ size (Zecca, 2010).

Differential requirement of Salvador-Warts-Hippo pathway members for organ size control in Drosophila melanogaster

The Salvador-Warts-Hippo (SWH) pathway contains multiple growth-inhibitory proteins that control organ size during development by limiting activity of the Yorkie oncoprotein. Increasing evidence indicates that these growth inhibitors act in a complex network upstream of Yorkie. This complexity is emphasised by the distinct phenotypes of tissue lacking different SWH pathway genes. For example, eye tissue lacking the core SWH pathway components salvador, warts or hippo is highly overgrown and resistant to developmental apoptosis, whereas tissue lacking fat or expanded is not. This study explores the relative contribution of SWH pathway proteins to organ size control by determining their temporal activity profile throughout Drosophila eye development. Eye tissue lacking fat, expanded or discs overgrown displays elevated Yorkie activity during the larval growth phase of development, but not in the pupal eye when apoptosis ensues. Fat and Expanded do possess Yorkie-repressive activity in the pupal eye, but loss of fat or expanded at this stage of development can be compensated for by Merlin. Fat appears to repress Yorkie independently of Dachs in the pupal eye, which would contrast with the mode of action of Fat during larval development. Fat is more likely to restrict Yorkie activity in the pupal eye together with Expanded, given that pupal eye tissue lacking both these genes resembles that of tissue lacking either gene. This study highlights the complexity employed by different SWH pathway proteins to control organ size at different stages of development (Milton, 2010).

The SWH pathway controls Drosophila eye size by limiting growth during the larval stage of development and by restricting proliferation and promoting apoptosis during pupal development. Eyes lacking core SWH pathway components (e.g. sav, wts or hpo) are significantly larger than eyes lacking the non-core components ft, ex, dco or Mer. Owing to this disparity, it has been hypothesized that ft and ex only partially affect SWH pathway activity, whereas sav, wts and hpo have stronger effects, or, alternatively, that non-core components affect pathway activity in a temporally restricted fashion. Analysis of tissue recessive for ft, ex or dco³ revealed that Yki activity was elevated during larval eye development when tissues are actively growing and proliferating, but not during pupal development when apoptosis ensues, supporting the idea that Ft, Ex and Dco influence SWH pathway activity in a temporally restricted fashion. However, when tissue lacking both Mer and ft, or Mer and ex, was analysed, Yki activity was found to be elevated during both larval and pupal development, similar to the Yki activity profile observed in tissue lacking core SWH pathway proteins. This is consistent with previous reports showing that Mer acts in parallel to both Ft and Ex, and that these proteins can compensate for each other to control SWH pathway activity. Therefore, Ft and Ex do contribute to SWH pathway regulation in the pupal eye to ensure appropriate exit from the cell cycle and developmental apoptosis, but these functions can be executed by Mer in their absence, suggesting a degree of plasticity in the regulation of Yki activity by non-core SWH pathway proteins. The ability of Mer to compensate for Ft or Ex cannot simply be explained by compensatory increases in Mer protein in pupal eye tissues lacking ft or ex, since Mer expression levels were found to be unaltered in these tissues (Milton, 2010).

Previous analyses of tissue lacking both ft and ex showed that these proteins function, at least in part, in parallel to control growth of larval imaginal discs. The current analysis of ft,ex double-mutant tissue suggests that these proteins are likely to function together to control Yki activity in the pupal eye. Yki activity was not elevated in tissue lacking ft, ex or both genes, showing that these genes cannot compensate for each other in the pupal eye. This is consistent with the notion that Ft influences the activity of downstream SWH pathway proteins by multiple mechanisms, an idea that is supported by THE analysis of the requirement of the atypical myosin, Dachs, for Ft signalling in the pupal eye. During larval imaginal disc development, Ft can influence Yki activity by repressing Dachs activity, which in turn can repress the core SWH pathway protein Wts. Analysis of pupal eye tissue that lacks both Mer and ft, or Mer, ft and dachs, showed that Yki activity was elevated in each scenario. This shows that in the pupal eye, the ability of Ft to compensate for Mer is not reliant on Dachs, and implies that Ft can employ different modes of signal transduction throughout eye development. However, because Ft and Mer can compensate for each other it is not possible to formally conclude that normal signal transduction by Ft in the pupal eye occurs independently of Dachs (Milton, 2010).

Expression of Ex is tightly controlled in response to alterations in SWH pathway activity at both the transcriptional and post-transcriptional levels. Interestingly, it was also found that Ex expression is controlled in a temporal fashion throughout eye development; Ex is expressed at relatively high levels in the larval eye, but at very low levels in the pupal eye. Despite the fact that Ex expression is very low in the pupal eye, it clearly retains function at this stage of development because it can compensate for loss of Mer to restrict Yki activity. The dynamic expression profile of Ex suggests that factors that influence its expression play an important role in defining overall eye size in Drosophila. At present, only two transcriptional regulatory proteins have been shown to influence the expression of ex: Yki and Sd. There are conflicting reports on whether Yki and Sd control basal expression of ex in larval imaginal discs. It is clear, however, that Yki and Sd collaborate to drive ex expression when the activity of the SWH pathway is suppressed, presumably as part of a negative-feedback loop. Despite the fact that basal ex expression is low in the pupal eye, the ex promoter is still responsive to Yki, as Ex expression is substantially elevated in pupal eye clones lacking hpo or Mer and ex. Future investigation of the ex promoter will help to clarify understanding of the complex fashion by which expression of the ex gene is controlled, and should aid understanding of eye size specification in Drosophila (Milton, 2010).

This study emphasises the complexity of the means by which the activity of core SWH pathway proteins is regulated by non-core proteins such as Ft, Ex, Mer and Dco. The signalling mechanisms employed by non-core proteins appear to differ at discrete stages of development in order to achieve appropriate organ size during the larval growth period of eye development, and to subsequently sculpt the eye by regulating apoptosis during pupal development (Milton, 2010).

Planar polarization of the atypical myosin Dachs orients cell divisions in Drosophila

Tissues can grow in a particular direction by controlling the orientation of cell divisions. This phenomenon is evident in the developing Drosophila wing epithelium, where the tissue becomes elongated along the proximal-distal axis. Orientation of cell divisions in the wing requires planar polarization of an atypical myosin, Dachs. Evidence suggests that Dachs constricts cell-cell junctions to alter the geometry of cell shapes at the apical surface and that cell shape then determines the orientation of the mitotic spindle. Using a computational model of a growing epithelium, it was shown that polarized cell tension is sufficient to orient cell shapes, cell divisions, and tissue growth. Planar polarization of Dachs is ultimately oriented by long-range gradients emanating from compartment boundaries, and is therefore a mechanism linking these gradients with the control of tissue shape (Mao, 2011).

Recently, Dachs was found to be localized in a planar-polarized manner along the P-D axis in response to the Dachsous gradient. This study observed that the localization of Dachs correlates with the orientation of cell divisions and tissue growth in the developing fly wing. Dachs localizes to the distal side of each cell's apical surface, and clones tend to grow preferentially along the P-D axis to form elongated shapes. This intriguing correlation has led to a speculation that Dachs might mediate the orientation of cell divisions and tissue growth in the fly wing (Mao, 2011).

To test this hypothesis, the behavior of marked clones of cells was examined in wing discs lacking functional Dachs protein (dachs mutant), and wing discs in which Dachs is abnormally localized around the entire apical cell surface (fat mutant). The normal elongation of clone shapes along the P-D axis is completely disrupted in either dachs mutant discs, with clones tending to be rounded and misoriented. Orientation of mitotic spindles is also disrupted in these mutants. This failure to orient cell divisions in dachs and fat mutants results in abnormally shaped adult wings that are reduced in the P-D axis relative to wild-type controls. This shape change is evident despite opposite effects of the two mutations on size. These results indicate that planar polarization of Dachs is essential for orienting cell divisions and tissue growth (Mao, 2011).

Whether reorientation of Dachs is sufficient to reorient cell divisions and tissue growth was examined. Dachsous was expressed with the dpp.Gal4 driver, which is expressed in a gradient along the A-P axis in the anterior compartment. This ectopic gradient of Dachsous runs perpendicular to the endogenous gradient and repolarizes Dachs. As a result, clones are reoriented perpendicular to the P-D axis, as are mitotic spindles. In adult wings, the ectopic Dachsous gradient drives elongation of the wing perpendicular to the P-D axis, in the anterior compartment (Mao, 2011).

How might Dachs control the orientation of the mitotic spindle? Dachs might directly orient the mitotic spindle by tethering. Alternatively, since Dachs is a myosin, it might indirectly orient the spindle by controlling cell shape. Existing evidence supports the latter view: Mitotic spindles align along the long axis of the cell prior to division in both yeast and mammalian cells. In the case of epithelial cells, spindles are restricted to the plane of the epithelium and their orientation may be affected by apical surface geometry. Apical cell shape and the orientation of cell division were examined in live wing discs in culture; cells were found to divide along their long apical axis. This correlation persists even in cases where divisions are not oriented along the P-D axis. These results indicate that the geometry of apical cell-cell junctions determines the orientation of cell division (Mao, 2011).

To test whether Dachs controls cell shape at the apical surface, clones of dachs or fat mutant cells were examined in an otherwise wild-type disc. Cells mutant for dachs are, on average, 60% more dilated in their apical surface area than wild type. Cells mutant for fat are, on average, 40% more constricted than neighboring wild-type cells. When fat mutant cells are also mutant for dachs, their apical surface area reverts to that of single dachs mutants. Overexpression of Dachs enhances constriction of fat mutant cells. These results show that Dachs exerts a contractile force on apical cell junctions. Since Dachs is normally planar-polarized, it would be predicted to constrict cell-cell junctions at the distal end of each cell and the proximal end of its neighbor. Measurement of tension indicates that distal (and proximal) cell-cell junctions are under more tension than others. Consequently, the cell must grow by lengthening its other cell-cell junctions, resulting in cell shape elongation in the P-D axis prior to division, which then orients the mitotic spindle. This force-driven model is supported by the fact that clones of cells mutant for dachs show normally oriented growth when surrounded by wild-type cells that are capable of nonautonomously exerting force on the mutant clone. Thus, Dachs appears to act by exerting mechanical force because its loss can be compensated by restoring these forces (Mao, 2011).

To further test the proposal, a computational model of a growing epithelial tissue was built that was adapted from a previous model. It was found that, compared with isometric apical tension, polarized apical tension is sufficient to orient cell divisions and tissue growth. As in vivo, cell divisions are only imperfectly correlated with the P-D axis in this model; this emergent behavior is in fact important to generate the type of clone shapes observed in the developing wing. If cell divisions are forcibly oriented in the P-D axis, abnormal clone shapes result and the link between cell shape and orientation of division is broken, indicating that Dachs is unlikely to directly orient the spindle in vivo. This model therefore supports the idea that Dachs indirectly orients the mitotic spindle by polarizing apical constriction to promote cell shape elongation in the P-D axis (Mao, 2011).

This model also explains an apparent paradox: Despite the presence of planar-polarized Dachs, the apical geometry of most wing cells is not elongated in the P-D axis. Indeed, elongation of cells in the P-D axis is commonly observed only in those cells about to undergo division. The simulations show that this is because elongated cells divide to produce daughter cells of more rounded or random shapes. The polarized tension exerted by Dachs is of moderate strength, and hence takes time to promote elongation in the P-D axis. Thus, only older cells ready to divide once more tend to show elongated forms. Furthermore, some cells never manage to elongate along the P-D axis, explaining why not all cell divisions occur in the P-D axis. The model shows how this complex behavior of cells in a tissue emerges simply from the mechanical properties of a dividing epithelium (Mao, 2011).

It is interesting to compare the function of the Dachs myosin in the proliferating wing epithelium with the function of myosin-II in convergent extension movements of the quiescent embryonic epithelium. Both myosins appear to generate anisotropic junctional tension, but at different strengths. In embryos, myosin-II is planar-polarized at gastrulation and exerts very strong tension, collapsing cell-cell junctions entirely such that cells rearrange their positions and intercalate. In the wing disc, Dachs appears much weaker than myosin-II because convergent extension movements are not obvious in live-imaged wing disc epithelia. Thus, orientation of cell divisions appears to require much more subtle polarization of cell tension than convergent extension, and this may explain why an atypical myosin, rather than myosin-II, orients cell divisions. It will be interesting to investigate the role of Dachs in other oriented cell division scenarios, such as wound healing (Mao, 2011).

In conclusion, planar polarization of Dachs links long-range gradients of secreted morphogens - known to be the fundamental organizers of tissue pattern, size, and shape -- with the orientation of cell divisions and tissue growth during development. The mechanism that are proposed (a gradient directing the planar polarization of a molecule, which then orients cell behavior) may prove to be a widespread mechanism for organizing tissue development (Mao, 2011).

Fat/Hippo pathway regulates the progress of neural differentiation signaling in the Drosophila optic lobe

A large number of neural and glial cell cell types differentiate from neuronal precursor cells during nervous system development. Two types of Drosophila optic lobe neurons, lamina and medulla neurons, are derived from the neuroepithelial (NE) cells of the outer optic anlagen. During larval development, epidermal growth factor receptor (EGFR)/Ras signaling sweeps the NE field from the medial edge and drives medulla neuroblast (NB) formation. This signal drives the transient expression of a proneural gene, lethal of scute, and its signal array is referred to as the 'proneural wave', since it is the marker of the EGFR/Ras signaling front. This study shows that the atypical cadherin Fat and the downstream Hippo pathways regulate the transduction of EGFR/Ras signaling along the NE field and, thus, ensure the progress of NB differentiation. Fat/Hippo pathway mutation also disrupts the pattern formation of the medulla structure, which is associated with the regulation of neurogenesis. A candidate for the Fat ligand, Dachsous is expressed in the posterior optic lobe, and its mutation was observed to cause a similar phenotype as fat mutation, although in a regionally restricted manner. It was also shown that Dachsous functions as the ligand in this pathway and genetically interacts with Fat in the optic lobe. These findings provide new insights into the function of the Fat/Hippo pathway, which regulates the ordered progression of neurogenesis in the complex nervous system (Kawamori, 2011).

The Fat/Hippo pathway has been known as a tumor suppressor pathway. This study and in the report of Reddy (2010), it was shown that the loss of Fat/Hippo signaling causes a delay of NB differentiation in the optic lobe. In contrast, dachs;ft double mutation, which is expected to stabilize the Fat/Hippo pathway, causes an advance of NB differentiation. This led to the question of how the Fat/Hippo pathway controls NB differentiation (Kawamori, 2011).

It has been reported that EGFR/Ras signaling is necessary and sufficient for NB induction, and its transduction is the driving force of the progress of the proneural wave. EGFR/Ras signaling sweeps the NE field through the gradual activation of Ras and its downstream EGF secretion by Rho. It was reasoned that this signal transduction is the target of the Fat/Hippo pathway in the control of NB differentiation. Indeed, ectopic expression of the EGFR/Ras signaling components Ras^V12 and rho was sufficient to induce NBs in the ft mutant background (Kawamori, 2011).

Which step of this cycling process does Fat/Hippo pathway mutation affect? Based on ectopic expression experiments, the Fat/Hippo pathway lies upstream of Ras and Rho, and it is expected to control the process from EGF transmission to Ras activation. The phenotypic difference produced by Ras^V12 and rho overexpression should be noted. When rho was expressed in the ft mutant background, several NE clones with abnormal morphology remained. This phenotype was not observed when Ras^V12 was expressed. In this model, Ras^V12 drives NB differentiation in Ras^V12-expressing cells in a cell-autonomous manner. In contrast, Rho activates Ras signaling in neighboring cells through the secretion of an EGF ligand. Based on these phenotypic differences, the Fat/Hippo pathway is expected to control the cell-to-cell EGF transmission, including its secretion, distribution or reception at the cell surface. This hypothesis is supported by the fact that several signaling components of the EGFR/Ras pathway, including Rho and EGFR, are localized to the apical side of epithelial tissues, and it is thought that this signal is transmitted along the apical side in epithelial tissues. It has also been reported that Fat/Hippo pathway mutations enhance the expression level of several apically localized molecules, such as aPKC, PatJ, Crumbs and E-cadherin. Thus, Fat/Hippo signaling targets could include unknown apical components that are involved in EGF transmission and this could account for the incomplete NB induction by rho overexpression. rho-expressing cells secret the EGF ligand, which diffuses in the NE surface, but Fat/Hippo pathway mutation would prevent its cell-to-cell transmission and subsequent EGFR/Ras pathway activation in the receiving cells. In this hypothesis, EGF transmission would be disturbed in the NE mutant for the Fat/Hippo pathway, causing the delay of proneural wave progress (Kawamori, 2011).

As an alternative hypothesis, the Fat/Hippo pathway could regulate signal transduction from the EGFR to Ras activation. If this is the case, the Fat/Hippo pathway regulates the intracellular signal transduction of the EGFR pathway. Many of the known targets of the Fat/Hippo pathway are components of growth regulatory, cell survival and cell adhesion molecules. There could be unknown targets that modulate other signaling pathways, including the EGFR pathway, and the NB differentiation defect would thus be caused by a failure in the activation of differentiation signals in the absence of Fat/Hippo signaling (Kawamori, 2011).

This study shows that the Fat/Hippo pathway mutation also affected the morphological character of the NE. Fat/Hippo pathway mutant clones were induced, and they often included NE tissue with a folded morphology and disrupted the medulla structure. The results showed that the Fat/Hippo pathway functions in the regulation of NB differentiation and in NE morphology are distinct, but the two functions could affect each other. The morphological defect of the NE could affect EGFR/Ras signal transduction. The possibility is discussed that the EGF ligand could be distributed along the apical membrane of the NE. The invagination of the apical membrane of the folded NE into the inner region could prevent EGF ligand signaling. There were clones with a normal NE morphology in which NB differentiation was delayed and, thus, morphological defects are not determinate, but they could promote the delay of NB induction (Kawamori, 2011).

How is the activity of the Fat/Hippo pathway regulated throughout the development of the optic lobe? Ft is a member of the cadherin family, and an extracellular molecule is expected to regulate its activity. Ds is a candidate for the Ft ligand that regulates planar cell polarity and Fat/Hippo signaling activity in other epithelial tissues. The expression of ds with a posterior-specific pattern in the developing optic lobe (Reddy, 2010) was confirmed. In the rescue experiments for the ds mutation, the expression of either ds lacking its intracellular domain (ds^ΔICD) or ft lacking its extracellular domain (ft^ΔECD) was sufficient to compensate for ds function, suggesting that Ds functions as a ligand and that Ft lies downstream of Ds in this context (Kawamori, 2011).

The phenotypes of ds and ft mutants were compared to assess whether the mutation of ds by itself accounts for the phenotype of the ft mutants. In contrast to the ft mutants that exhibited altered NB differentiation in the entire outer optic anlagen, the ds mutant phenotype was regionally specific; NB differentiation was severely delayed in the posterior region, and the development of the anterior region was not significantly affected. These differences suggest that there might be some regulatory mechanisms that control Ft activity independently of Ds in the anterior region of the optic lobe (Kawamori, 2011).

The Fat/Hippo pathway is known as a tumor suppressor pathway, and many studies related to this pathway have focused on tissue growth or cell survival. This study has reported a new function of the Fat/Hippo pathway in the regulation of neural differentiation. The Fat/Hippo pathway regulates the progress of neural differentiation signaling, and the EGFR/Ras pathway is a candidate target of this pathway. The data suggest that the Fat/Hippo pathway includes unknown targets involved in EGFR/Ras signal transduction. Further studies are required to identify the targets of the Fat/Hippo pathway and determine the interplay between Fat/Hippo and EGFR/Ras pathways, specifically in NB differentiation (Kawamori, 2011).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Regulation of cytoskeletal organization and junctional remodeling by the atypical cadherin Fat

The atypical cadherin Fat is a conserved regulator of planar cell polarity, but the mechanisms by which Fat controls cell shape and tissue structure are not well understood. This study shows that Fat is required for the planar polarized organization of actin denticle precursors, adherens junction proteins and microtubules in the epidermis of the late Drosophila embryo. In wild-type embryos, spatially regulated cell-shape changes and rearrangements organize cells into highly aligned columns. Junctional remodeling is suppressed at dorsal and ventral cell boundaries, where adherens junction proteins accumulate. By contrast, adherens junction proteins fail to accumulate to the wild-type extent and all cell boundaries are equally engaged in junctional remodeling in fat mutants. The effects of loss of Fat on cell shape and junctional localization, but not its role in denticle organization, are recapitulated by mutations in Expanded, an upstream regulator of the conserved Hippo pathway, and mutations in Hippo and Warts, two kinases in the Hippo kinase cascade. However, the cell shape and planar polarity defects in fat mutants are not suppressed by removing the transcriptional co-activator Yorkie, suggesting that these roles of Fat are independent of Yorkie-mediated transcription. The effects of Fat on cell shape, junctional remodeling and microtubule localization are recapitulated by expression of activated Notch. These results demonstrate that cell shape, junctional localization and cytoskeletal planar polarity in the Drosophila embryo are regulated by a common signal provided by the atypical cadherin Fat and suggest that Fat influences tissue organization through its role in polarized junctional remodeling (Marcinkevicius, 2013).

Fat regulates planar polarity in many cells and tissues, but the mechanisms by which Fat influences cell and tissue structure are not well understood. This study shows that Fat is required for the localization of denticle actin precursors, microtubules and adherens junction proteins in the Drosophila embryo. Junctional remodeling in wild-type embryos is selectively suppressed at dorsal and ventral cell borders, which are sites of increased adherens junction accumulation. In fat mutants, decreased junctional planar polarity is associated with excessive growth and contraction at dorsal and ventral cell borders and a reduction in cell elongation and alignment. These results suggest a model in which Fat regulates planar polarized junctional localization and remodeling, which in turn influences cell shape. The atypical myosin Dachs is asymmetrically localized in denticle-forming cells and loss of dachs partially suppresses the denticle and junctional defects in fat mutants, suggesting that aberrant Dachs activity contributes to the defects in fat mutants. However, junctional and denticle planar polarity are correctly established in dachs mutants, indicating that Dachs is not essential for these processes. Defects in junctional planar polarity, but not denticle localization, are recapitulated in embryos that express activated Notch and embryos mutant for Expanded, Hippo or Warts. These results demonstrate an essential role for Fat and Hippo/Warts signaling in regulating planar polarized adherens junction localization in the Drosophila embryo (Marcinkevicius, 2013).

Although the Hippo/Warts pathway has generally been thought to be separate from the role of Fat in planar polarity, this study demonstrates an unexpected role for Expanded, Hippo and Warts in junctional planar polarity in the Drosophila embryo. Hippo/Warts signaling could influence planar polarity by modulating the size of the apical epithelial domain or the levels of junctional and signaling proteins or by altering the expression of Four-jointed, a kinase that regulates the interaction between Fat and Ds. However, these effects all require transcriptional activation by Yorkie. By contrast, the junctional defects in fat mutants are not suppressed by removing Yorkie or recapitulated by Yorkie activation, indicating that the role of Fat in planar polarity is independent of Yorkie-mediated transcription. Yorkie-independent effects of the Hippo/Warts pathway regulate dendrite maintenance and apical actin levels in epithelia. These results raise the possibility that other effectors of Expanded, or other substrates of Warts, could mediate the effects of Fat on junctional planar polarity directly through signaling events at the cell cortex (Marcinkevicius, 2013).

Junctional planar polarity in the denticle field is distinct from cytoskeletal planar polarity in several ways, suggesting that these processes involve different mechanisms of Fat signaling. Like hairs in the Drosophila wing, denticles are unipolar structures that initiate from the posterior cell cortex. By contrast, adherens junctions are homophilic complexes that are likely to accumulate at both dorsal and ventral cell boundaries in a bipolar fashion. Second, denticle defects are specific to the posterior denticle belt in fat and ds mutants, whereas defects in cell shape, junctional localization and microtubule organization occur more broadly. Third, the placement of denticle actin precursors occurs normally, despite defects in cell shape and junctional localization, in activated Notch-expressing embryos and ex, hippo or warts mutants, demonstrating that these processes have distinct molecular requirements. In one model, Fat could regulate different processes by signaling through different effectors, with Ex, Hippo and Warts acting specifically in junctional regulation. Alternatively, Fat could influence different structures by acting at different locations in the cell. Further studies of the effectors required for the role of Fat in planar polarity will provide insight into the mechanisms by which Fat regulates multiple aspects of cellular organization (Marcinkevicius, 2013).

The similar defects in fat mutants and embryos that express activated Notch suggest that Fat and Notch might affect a common process regulating cell shape and polarity. Activated Notch could disrupt cell shape and polarity indirectly, perhaps through changes in epidermal growth factor (EGF) receptor signaling. Notch has been shown to be involved in morphogenetic processes such as compartment boundary formation in the Drosophila wing, and an ectopic stripe of Notch is sufficient to induce cell alignment. The current results raise the possibility that Notch could influence cell morphology through its role in microtubule or junctional organization (Marcinkevicius, 2013).

This study shows that Fat is required for the organization of cells into aligned columns with discrete identities. Junctional remodeling in the denticle field is distinct from other examples of polarized cell behavior. During axis elongation in the Drosophila embryo, spatially regulated actomyosin contractility promotes junctional disassembly and inhibits junctional assembly at cell boundaries perpendicular to the axis of tissue elongation. Despite a similar localization of myosin in the denticle field, most shrinking and growing edges share the same orientation. These results suggest a novel form of polarized junctional remodeling in which junctional assembly and disassembly are activated in the same cellular domain, whereas other domains are largely quiescent. These behaviors could provide a mechanism that allows cells to maintain an unusual elongated, highly aligned configuration by continually exerting tension on each other's boundaries, resulting in an inevitable degree of edge growth and contraction. The results suggest that in addition to polarized actomyosin contractility, junctional remodeling must be actively suppressed at dorsal and ventral cell boundaries in a Fat-dependent mechanism that is necessary for cell shape and tissue organization. This mechanism may be generally relevant to other structures that form during compartment boundary formation, ectoderm segmentation, and lens and retinal cells in the vertebrate eye. Fat/Dachsous signaling is required for polarized cell division and cell rearrangements in Drosophila, and for tissue elongation in the mouse neural tube, cochlea, kidney and intestines. The ability of Fat to regulate junctional remodeling and cytoskeletal polarity may be important for its diverse roles in tissue organization (Marcinkevicius, 2013).

Local cell death changes the orientation of cell division in the developing Drosophila wing imaginal disc without using Fat or Dachsous as orienting signals

Drosophila imaginal disc cells exhibit preferred cell division orientations according to location within the disc. These orientations are altered if cell death occurs within the epithelium, such as is caused by cell competition or by genotypes affecting cell survival. Both normal cell division orientations, and their orientations after cell death, depend on the Fat-Dachsous pathway of planar cell polarity (PCP). The hypothesis that cell death initiates a planar polarity signal was investigated. When clones homozygous for the pineapple eye (pie) mutation were made to initiate cell death, neither Dachsous nor Fat was required in pie cells for the re-orientation of nearby cells, indicating a distinct signal for this PCP pathway. Dpp and Wg were also not needed for pie clones to re-orient cell division. Cell shapes were evaluated in wild type and mosaic wing discs to assess mechanical consequences of cell loss. Although proximal wing disc cells and cells close to the dorso-ventral boundary were elongated in their preferred cell division axes in wild type discs, cell shapes in much of the wing pouch were symmetrical on average and did not predict their preferred division axis. Cells in pie mutant clones were slightly larger than their normal counterparts, consistent with mechanical stretching following cell loss, but no bias in cell shape was detected in the surrounding cells. These findings indicate that an unidentified signal influences PCP-dependent cell division orientation in imaginal discs (Kale, 2016).

This paper made use of the observation that clones of imaginal disc cells mutant for pie, which exhibit an elevated rate of apoptosis, bias the cell division orientation of other cells nearby in a search for a signal responsible for cell division orientation. It is hypothesized that dying pie cells may be the source of a polarizing signal that is detected by other cells, and the roles of candidate signals were evaluated by removing them genetically from pie mutant cells. It is further hypothesized that the result may also be relevant to the orientation of cell divisions in normal development (Kale, 2016).

Since cell division orientation requires the PCP receptor Fat, this study tested whether its PCP ligand Dachsous was required, but this model was excluded. Since cell division orientation also requires Dachsous in the dividing cells, tests were performed to see whether Fat itself was a signal required in the apoptotic clones, but this was also excluded. In fact both Fat and Dacshous could be eliminated together from the dying cell population without preventing the orientation of nearby cells. The possibility was considered that rather than expressing Fat or Dachsous, apoptotic cells might down-regulate one or both proteins and that this might affect nearby cells, but it was found that eliminating one or both genes was not sufficient to orient nearby cell divisions. The possible contribution of Four-jointed, a kinase that phosphorylates Fat and Dachsous proteins in the Golgi, was not tested because Four-jointed should be unable to signal in cells already mutated for both ft and ds. Altogether, the experiments eliminated known ligands for the Fat/Dachsous PCP pathway, suggesting that the pathway must be required to orient cell division in response to some other signal (Kale, 2016).

It has been suggested that apoptotic imaginal disc cells secrete the morphogens Dpp and Wg in the process of stimulating compensatory proliferation. Since Dpp and Wg pattern many aspects of imaginal disc development, including the expression of some PCP genes, they were candidates to orient the division of imaginal disc cells. Contrary to this prediction, clones of apoptotic cells lacking Dpp and Wg continued to orient nearby cell divisions. It cannot be excluded that there may be other biochemical signals from dying cells that orient cell division. For example, there are other Wnt proteins in Drosophila that might affect cell polarity (Kale, 2016).

One other model consistent with these results is that cell division is oriented by physical constraints rather than biochemical signals. It is thought that in the wild type wing disc, the characteristic circumferential division pattern of the peripheral cells is a result of their being stretched around the growing wing pouch. Consistent with this conclusion, it has been reported that when a clone of cells grows more rapidly than the surrounding epithelium, cells around the clone are stretched circumferentially to accommodate the hyperplastic region, and this change in shape tends to orient cell divisions in a circumferential pattern around the hyperplastic clones. By analogy to these findings concerning enhanced growth, it might be expected that clones of cells experiencing high rates of cell death would expand more slowly than surrounding cells, and that this would stretch the cells around the clone inwards towards the slow growing region, leading to a reorientation of cell divisions towards the slow growing clone, opposite to the case of more rapidly growing clones. As expected given their persistent cell death, clones of pie homozygous cells grow more slowly than control clones, and exhibit a small increase in apical cell size, consistent with local tension in the epithelium. The changed orientation of cell division near to pie clones has been reported previously. This study was unable, however, to measure a consistent change in shape of the wing cells adjacent to pie homozygous clones, the population of cells where the altered division orientation is measured. This lack of correlation between cell shape and cell division orientation is also seen for wing pouch cells in the wild type wing disc, which show a proximo-distal division preference but no obvious proximo-distal polarization. The shapes of mitotic cells were not measured separately, and so the possibility cannot be excluded that only the mitotic cells exhibit altered shapes in the wing pouch. Recently, it has been reported that the orientation of epithelial cell division is determined by microtubule interactions with cell junction vertices, and that cell shape is a poor predictor of cell division in rounded cells, where the disposition of cell junction vertices varies. This may explain why both the normal cell division orientation and the response to cell death do not correlate with cell shape within the wing pouch region, where cells are more rounded than in peripheral regions of the wing disc (Kale, 2016).

Oriented cell divisions are suggested to contribute to organogenesis. It was suggested that oriented cell divisions are responsible for the shape of cell clones in the wing disc, which ultimately determines the shape of the whole tissue (which is a collection of clones). Oriented cell divisions may have other functions, for example they may represent a homeostatic mechanism that ameliorates growth-induced mechanical stress (Kale, 2016).

The shape of cell clones becomes less regular during cell competition, and the interfaces between wild type and Minute cell populations become more convoluted and interdigitated. Previously, it was suggested that oriented cell division could be responsible for the intermingling of wild type and Minute cells. Recently, Levayer described very similar intermingling between cells in the pupal notum that are induced to compete by expression of different levels of Myc protein (Levayer, 2015). Very little cell division occurs in pupal notum, and Levayer describe cell neighbor exchanges that are responsible for intermingling the cell populations. They propose these exchanges are promoted by mechanical effects of differential growth rates. Wild type and Minute cells also grow at different rates, but the apoptotic protein baculovirus p35 reduces the degree of intermixing between wild type and Minute cells. There is now evidence that p35 also stimulates Minute growth rate, while having less effect on wild type cells. Although the precise mechanism is unclear, Minute cell growth is possibly stimulated by signals from the undead Rp/Rp cells that are preserved when p35 is expressed. Together these data raise the possibility that p35 may affect both cell division orientation and intermingling of wild type and Minute cells by equalizing their relative growth rates. In the case of pie clones that expand slowly, differential growth might result in local mechanical stretching which influences nearby cell divisions, although it cannot be excluded that the pie mutant clones have other differences from wild type (Kale, 2016).

Fat has a role as an upstream regulator of the Salvador-Warts-Hippo (SWH) pathway of tumor suppressors. There is substantial evidence that the SWH pathway responds to mechanical cues. Inputs are reported from actin polymerization status and from adhesion junctions via α-catenin and Juba proteins. Recent studies indicate that the SWH pathway itself promotes epithelial junctional tension, which is reduced in clones of ft or wts mutants. Cell division orientation also depends on atro, however, which has been thought not to affect SWH activity, since it does not affect growth. Recent studies suggest that mutations in the Fat-Dachsous pathway may affect PCP through a disruption of the Spiny Leg protein by de-repressed Dachs that is not a reflection of normal Dachs function. This does not explain how cell division orientation is affected by Fat or Dachsous but it does raise the possibility that Fat and Dachsous mutations might affect processes that depend little on their normal alleles. What this study reports is that the model developed for planar cell polarity, in which ligand-receptor interactions between Fat and gradients of Dachsous control cell polarity, do not seem applicable to the orientation of cell division in the wing disc, where mechanical factors may be important (Kale, 2016).

EVOLUTIONARY HOMOLOGS

The fat-like gene of Drosophila is the true orthologue of vertebrate fat cadherins and is involved in the formation of tubular organs

Fat cadherins constitute a subclass of the large cadherin family characterized by the presence of 34 cadherin motifs. To date, three mammalian Fat cadherins have been described; however, only limited information is known about the function of these molecules. This paper describes the second fat cadherin in Drosophila, fat-like (ftl). ftl is the true orthologue of vertebrate fat-like genes, whereas the previously characterized tumor suppressor cadherin, fat, is more distantly related as compared with ftl. Ftl is a large molecule of 4705 amino acids. It is expressed apically in luminal tissues such as trachea, salivary glands, proventriculus, and hindgut. Silencing of ftl results in the collapse of tracheal epithelia giving rise to breaks, deletions, and sac-like structures. Other tubular organs such as proventriculus, salivary glands, and hindgut are also malformed or missing. These data suggest that Ftl is required for morphogenesis and maintenance of tubular structures of ectodermal origin and underline its similarity in function to a reported lethal mouse knock-out of fat1 where glomerular epithelial processes collapse. Based on these results, a model is proposed where Ftl acts as a spacer to keep tubular epithelia apart rather than the previously described adhesive properties of the cadherin superfamily (Castillejo-Lopez, 2004).

Mammalian Fat genes

FAT, a new member of the human cadherin super-family, has been isolated from the T-leukemia cell line J6. The predicted protein closely resembles the Drosophila tumor suppressor Fat, which is essential for controlling cell proliferation during Drosophila development. The gene has the potential to encode a large transmembrane protein of nearly 4600 residues with 34 tandem cadherin repeats, five EGF-like repeats, and a laminin A-G domain. The cytoplasmic sequence contains two domains with distant homology to the cadherin catenin-binding region. Northern blotting analysis of J6 mRNA has demonstrated a full-length, approximately 15-kb, FAT message in addition to several 5'-truncated transcripts. In addition to its presence in J6 cells, in situ hybridization has revealed FAT mRNA expression in epithelia and in some mesenchymal compartments. Furthermore, higher levels of expression were observed in fetal, as opposed to adult, tissue, suggesting that its expression may be developmentally regulated in these tissues. FAT shows homologies with a number of proteins important in developmental decisions and cell:cell communication and is the first Fat-like protein reported in vertebrates. The gene encoding FAT was located by in situ hybridization on chromosome 4q34-q35. It is proposed that this family of molecules is likely to be important in mammalian developmental processes and cell communication (Dunne, 1995).

The expression during rat embryogenesis of the protocadherin fat, the murine homolog of a Drosophila tumor suppressor gene, has been examined. The sequence encodes a large protocadherin with 34 cadherin repeats, five epidermal growth factor (EGF)-like repeats containing a single laminin A-G domain and a putative transmembrane portion followed by a cytoplasmic sequence. This cytoplasmic sequence shows homology to the ß-catenin binding regions of classical cadherin cytoplasmic tails and also ends with a PDZ domain-binding motif. In situ hybridization studies at E15 show that fat is predominately expressed in fetal epithelial cell layers and in the CNS, although expression is also seen in tongue musculature and condensing cartilage. Within the CNS, expression is seen in the germinal regions and in areas of developing cortex, and this neural expression pattern is also seen at later embryonic (E18) and postnatal stages. No labelling was seen in adult tissues except in the CNS, where the remnant of the germinal zones, as well as the dentate gyrus, continue to express fat (Ponassi, 1999).

The entire sequence of the mouse Fat ortholog (mFat1) is presented. mFat1 protein consists of 4,588 amino acids with 34 cadherin repeats, 27 potential N-glycosylation sites, five EGF repeats and a laminin A G-motif in its extracellular domain. A single transmembrane region is followed by a cytoplasmic domain containing putative catenin-binding sequences. mFat1 shows high homology to human FAT and lesser homology to Drosophila Fat. The sequence of this giant cadherin suggests that it is unlikely to have a homophilic adhesive function, but may mediate heterophilic adhesion or play a signaling role. Expression analysis shows that the mfat1 gene is expressed early in pre-implantation mouse development, at the compact eight cell stage. Whole-mount and section in situ analyses show that transcripts are widely expressed throughout post-implantation development, most notably in the limb buds, branchial arches, forming somites, and in particular in the proliferating ventricular zones in the brain, being down-regulated as cells cease dividing. RT-PCR detects widespread expression in the adult, suggesting a role in proliferation and differentiation of many tissues and cell types (Cox, 2000).

Slit diaphragms are intercellular junctions of podocytes of the renal glomerulus. The molecular composition of slit diaphragms is still elusive. Slit diaphragms are characterized by the presence of a wide intercellular space. The morphological feature is shared by desmosomes and adherens junctions, which contain members of the cadherin superfamily. Thus, it has been hypothesized that some components of slit diaphragms belong to the cadherin superfamily. Consequently, cDNA encoding FAT has been isolated from reverse-transcribed (RT) glomerular cDNA by homology polymerase chain reaction (PCR) using primers based on conserved sequences in cadherin molecules. FAT is a novel member of the cadherin superfamily with 34 tandem cadherin-like extracellular repeats, and it closely resembles the Drosophila tumor suppressor fat. Expression of FAT was examined in glomeruli of the adult rat kidney by the ribonuclease protection assay and in situ hybridization. To localize the FAT protein in podocytes minutely, affinity-purified antibody was prepared against FAT by immunizing rabbits against an oligopeptide corresponding to the C-terminal 20 amino acids. Expression of FAT mRNA was detected in total RNA from glomeruli. In situ hybridization revealed significant signals in podocytes. Western blot analysis using solubilized glomeruli showed a single band, in which the molecular weight was more than 500 kD. Immunostaining of cultured epithelial cells from rat kidney (NRK52E) revealed FAT accumulation in cell-cell contact sites. In the glomerulus, FAT staining is observed distinctly along glomerular capillary walls. Double-label immunostaining using monoclonal antibody against slit diaphragms (mAb 5-1-6) showed identical localization of anti-FAT antibody and mAb 5-1-6. Furthermore, the double-label immunogold technique with ultrathin cryosections demonstrates that gold particles for FAT cytoplasmic domain are located at the base of slit diaphragms labeled by mAb 5-1-6 and that the cytoplasmic domain of FAT colocalizes with ZO-1, a cytoplasmic component associated with slit diaphragms. Thus the molecular structure of FAT and its colocalization with 5-1-6 antigen and ZO-1 indicate that FAT is a component of slit diaphragms (Inoue, 2001).

Using computer-based, motif-trap screening, a third member of the mammalian fat family, fat3, has been identified. Human and rat fat3 are also similar to the Drosophila tumor suppressor gene fat. The rat fat3 gene encodes a large protein of 4555 amino acids with 34 cadherin domains, 4 epidermal growth factor (EGF)-like motifs, a laminin A-G motif, and a cytoplasmic domain. Each member of the fat family is differentially expressed in the central nervous system during development. While both fat3 mRNA and fat1 mRNA are abundantly expressed in the fetal brain, the expression of MEGF1/fat2 mRNA is restricted to the postnatal cerebellum. fat3 mRNA and protein expression in the brain peaks at E15 during embryonic development. During this time, robust fat3 immunoreactivity is also observed in the spinal cord. These data suggest that the fat3 protein plays an important role in axon fasciculation and modulation of the extracellular space surrounding axons during embryonic development (Mitsui, 2002).

Cell migration requires integration of cellular processes resulting in cell polarization and actin dynamics. Previous work using tools of Drosophila genetics suggested that protocadherin Fat serves in a pathway necessary for determining cell polarity in the plane of a tissue. This study identifies mammalian FAT1 as a proximal element of a signaling pathway that determines both cellular polarity in the plane of the monolayer and directs actin-dependent cell motility. FAT1 is localized to the leading edge of lamellipodia, filopodia, and microspike tips where FAT1 directly interacts with Ena/VASP proteins that regulate the actin polymerization complex. When targeted to mitochondrial outer leaflets, FAT1 cytoplasmic domain recruits components of the actin polymerization machinery sufficient to induce ectopic actin polymerization. In an epithelial cell wound model, FAT1 knockdown decreases recruitment of endogenous VASP to the leading edge and results in impairment of lamellipodial dynamics, failure of polarization, and an attenuation of cell migration. FAT1 may play an integrative role regulating cell migration by participating in Ena/VASP-dependent regulation of cytoskeletal dynamics at the leading edge and by transducing an Ena/VASP-independent polarity cue (Moeller, 2004).

Fat cadherins form a distinct subfamily of the cadherin gene superfamily and are featured by their unusually large extracellular domain. This work investigated the function of a mammalian Fat cadherin. Fat1 is localized at filopodial tips, lamellipodial edges, and cell-cell boundaries, overlapping with dynamic actin structures. RNA interference-mediated knockdown of Fat1 results in disorganization of cell junction-associated F-actin and other actin fibers/cables, disturbance of cell-cell contacts, and also inhibition of cell polarity formation at wound margins. Furthermore, Ena/vasodilator-stimulated phosphoproteins were identified as a potential downstream effector of Fat1. These results suggest that Fat1 regulates actin cytoskeletal organization at cell peripheries, thereby modulating cell contacts and polarity (Tanoe, 2004).

The significance of cadherin superfamily proteins in vascular smooth muscle cell (VSMC) biology is undefined. Recent studies of the Fat1 protocadherin show that expression in VSMCs increases significantly after arterial injury or growth factor stimulation. Fat1 knockdown decreases VSMC migration in vitro, but surprisingly, enhances cyclin D1 expression and proliferation. Despite limited similarity to classical cadherins, the Fat1 intracellular domain (Fat1_IC) interacts with beta-catenin, inhibiting both its nuclear localization and transcriptional activity. Fat1 undergoes cleavage and Fat1_IC species localize to the nucleus; however, inhibition of the cyclin D1 promoter by truncated Fat1_IC proteins corresponds to their presence outside the nucleus, which argues against repression of beta-catenin-dependent transcription by nuclear Fat1_IC. These findings extend recent observations about Fat1 and migration in other cell types, and demonstrate for the first time its anti-proliferative activity and interaction with beta-catenin. Because it is induced after arterial injury, Fat1 may control VSMC functions central to vascular remodeling by facilitating migration and limiting proliferation (Hou, 2006).

The cadherin superfamily protein Fat1 is known to interact with the EVH1 domain of mammalian Ena/VASP. This study demonstrates that the scaffolding proteins Homer-3 and Homer-1 also interact with the EVH1 binding site of hFat1 in vitro; binding of Homer-3 and Mena to hFat1 is mutually competitive. Endogenous Fat1 binds to immobilised Homer-3 and endogenous Homer-3 binds to immobilised Fat1. Both, endogenous and over-expressed Fat1 exhibit co-localisation with Homer-3 in cellular protrusions and at the plasma membrane of HeLa cells. Since Homer proteins and Fat1 have been both linked to psychic disorders, their interaction may be of patho-physiological importance (Schreiner, 2006).

Functional interactions between Fat family cadherins in tissue morphogenesis and planar polarity

The atypical cadherin fat (ft) was originally discovered as a tumor suppressor in Drosophila and later shown to regulate a form of tissue patterning known as planar polarity. In mammals, four Ft homologs have been identified (Fat1-4). Recently, it has been demonstrated that Fat4 plays a role in vertebrate planar polarity. Fat4 has the highest homology to ft, whereas other Fat family members are homologous to the second ft-like gene, ft2 (kugelei). Genetic studies in flies and mice imply significant functional differences between the two groups of Fat cadherins. This study demonstrates that Fat family proteins act both synergistically and antagonistically to influence multiple aspects of tissue morphogenesis. Fat1 and Fat4 cooperate during mouse development to control renal tubular elongation, cochlear extension, cranial neural tube formation and patterning of outer hair cells in the cochlea. Similarly, Fat3 and Fat4 synergize to drive vertebral arch fusion at the dorsal midline during caudal vertebra morphogenesis. Evidence is provided that these effects depend on conserved interactions with planar polarity signaling components. In flies, the transcriptional co-repressor Atrophin (Atro) physically interacts with Ft and acts as a component of Fat signaling for planar polarity. The mammalian orthologs of atro, Atn1 and Atn2l, modulate Fat4 activity during vertebral arch fusion and renal tubular elongation, respectively. Moreover, Fat4 morphogenetic defects are enhanced by mutations in Vangl2, a 'core' planar cell polarity gene. These studies highlight the wide range and complexity of Fat activities and suggest that a Fat-Atrophin interaction is a conserved element of planar polarity signaling (Saburi, 2012).

Fat cadherins comprise a diverse family of proteins with substantially different intracellular domains; this study has shown that some function redundantly to pattern tissues, whereas others function antagonistically. Moreover, Fat family members exhibit tissue-specific interactions, such as Fat1 and Fat4 in kidney, cochlea, cranial neural tube and Fat3 and Fat4 in vertebra. Although several Fat loss-of-function phenotypes in mice reflect altered planar polarity signaling, such as cystic kidneys and shortened cochleae, other phenotypes may not be due to defective planar polarity and might instead reflect altered growth regulation. Consistent with this model, only a subset of Fat4^-/- phenotypes is enhanced by disrupted Vangl2 activity. The data also suggest that atrophins might function with Fat cadherins in vertebrates, as in flies, to regulate planar polarity. Finally, strong inbred genetic background effects have been seen in Fat loss-of-function phenotypes, not only in the cranial neural tube, which generally shows a clear strain dependency of defects, but also in other tissues. These as yet unidentified modifiers might impact the distinct phenotypic readouts caused by loss of Fat, such as defective Fat-planar polarity signaling versus Fat-Hippo signaling. Although these complexities introduce new challenges, the current findings emphasize the wide-ranging impact that Fat cadherins have on animal development (Saburi, 2012).

Regulation of neuronal migration by Dchs1-Fat4 planar cell polarity

Planar cell polarity (PCP) describes the polarization of cell structures and behaviors within the plane of a tissue. PCP is essential for the generation of tissue architecture during embryogenesis and for postnatal growth and tissue repair, yet how it is oriented to coordinate cell polarity remains poorly understood. In Drosophila, PCP is mediated via the Frizzled-Flamingo (Fz-PCP) and Dachsous-Fat (Fat-PCP) pathways. Fz-PCP is conserved in vertebrates, but an understanding in vertebrates of whether and how Fat-PCP polarizes cells, and its relationship to Fz-PCP signaling, is lacking. Mutations in human FAT4 and DCHS1, key components of Fat-PCP signaling, cause Van Maldergem syndrome, characterized by severe neuronal abnormalities indicative of altered neuronal migration. This study investigated the role and mechanisms of Fat-PCP during neuronal migration using the murine facial branchiomotor (FBM) neurons as a model. Fat4 and Dchs1 were found to be expressed in complementary gradients and are required for the collective tangential migration of FBM neurons and for their PCP. Fat4 and Dchs1 are required intrinsically within the FBM neurons and extrinsically within the neuroepithelium. Remarkably, Fat-PCP and Fz-PCP regulate FBM neuron migration along orthogonal axes. Disruption of the Dchs1 gradients by mosaic inactivation of Dchs1 alters FBM neuron polarity and migration. This study implies that PCP in vertebrates can be regulated via gradients of Fat4 and Dchs1 expression, which establish intracellular polarity across FBM cells during their migration. The results also identify Fat-PCP as a novel neuronal guidance system and reveal that Fat-PCP and Fz-PCP can act along orthogonal axes (Zakaria, 2014).

Fat3 and Ena/VASP proteins influence the emergence of asymmetric cell morphology in the developing retina

Neurons exhibit asymmetric morphologies throughout development - from migration to the elaboration of axons and dendrites - that are correctly oriented for the flow of information. For instance, retinal amacrine cells migrate towards the inner plexiform layer (IPL) and then retract their trailing processes, thereby acquiring a unipolar morphology with a single dendritic arbor restricted to the IPL. This study provides evidence that the Fat-like cadherin Fat3 (see Drosophila Fat) acts during multiple stages of amacrine cell development in mice to orient overall changes in cell shape towards the IPL. Using a time-lapse imaging assay, this study found that developing amacrine cells are less directed towards the IPL in the absence of Fat3, during both migration and retraction. Consistent with its predicted role as a cell-surface receptor, Fat3 functions cell-autonomously and is able to influence the cytoskeleton directly through its intracellular domain, which can bind and localize Ena/VASP family (see Drosophila Enabled) actin regulators. Indeed, a change in Ena/VASP protein distribution is sufficient to recapitulate the Fat3 mutant amacrine cell phenotype. Thus, Fat-like proteins might control the polarized development of tissues by sculpting the cytoskeleton of individual cells (Krol, 2016).

REFERENCES

Search PubMed for articles about Drosophila fat

Alagramam, K. N., et al. (2001). The mouse Ames waltzer hearing-loss mutant is caused by mutation of Pcdh15, a novel protocadherin gene. Nat. Genet. 27: 99-102. 11138007

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Baena-López, L. A., Baonza, A. and García-Bellido, A. (2005). The orientation of cell divisions determines the shape of Drosophila organs. Curr. Bio. 15: 1640-1644. 16169485

Baena-Lopez, L. A., Rodriguez, I. and Baonza, A. (2008). The tumor suppressor genes dachsous and fat modulate different signalling pathways by regulating dally and dally-like. Proc Natl Acad Sci U S A 105: 9645-9650. PubMed ID: 18621676

Bando, T., Mito, T., Maeda, Y., Nakamura, T., Ito, F., Watanabe, T., Ohuchi, H. and Noji, S. (2009). Regulation of leg size and shape by the Dachsous/Fat signalling pathway during regeneration. Development 136(13): 2235-45. PubMed Citation: 19474149

Bossuyt, W., Chen, C. L., Chen, Q., Sudol, M., McNeill, H., Pan, D., Kopp, A. and Halder, G. (2014). An evolutionary shift in the regulation of the Hippo pathway between mice and flies. Oncogene 33: 1218-1228. PubMed ID: 23563179

Bosch, J. A., Sumabat, T. M., Hafezi, Y., Pellock, B. J., Gandhi, K. D. and Hariharan, I. K. (2014). The Drosophila F-box protein Fbxl7 binds to the protocadherin Fat and regulates Dachs localization and Hippo signaling. Elife 3: e03383. PubMed ID: 25107277

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Buratovich, M. A. and Bryant, P. J. (1997). Enhancement of overgrowth by gene interactions in lethal(2)giant discs imaginal discs from Drosophila melanogaster. Genetics 147(2): 657-670. PubMed Citation: 9335602

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Castillejo-Lopez, C., Arias, W. M. and Baumgartner, S. (2004). The fat-like gene of Drosophila is the true orthologue of vertebrate fat cadherins and is involved in the formation of tubular organs. J. Biol. Chem. 279(23): 24034-43. 15047711

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

Cox, B., Hadjantonakis, A. K., Collins, J. E. and Magee, A. I. (2000). Cloning and expression throughout mouse development of mfat1, a homologue of the Drosophila tumour suppressor gene fat. Dev. Dyn. 217(3): 233-40. 10741417

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Dunne, J., et al. (1995). Molecular cloning and tissue expression of FAT, the human homologue of the Drosophila fat gene that is located on chromosome 4q34-q35 and encodes a putative adhesion molecule. Genomics 30(2): 207-23. 8586420

Fanto, M., et al. (2003). The tumor-suppressor and cell adhesion molecule Fat controls planar polarity via physical interactions with Atrophin, a transcriptional co-repressor. Development 130: 763-774. 12506006

Aigouy, B., et al. (2010). Cell flow reorients the axis of planar polarity in the wing epithelium of Drosophila. Cell 142: 773-786. PubMed Citation: 20813263

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