See the embryonic expression pattern of ft at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.
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).
In the Drosophila wing, distal cells signal to proximal cells to induce the expression of Wingless, but the basis for this distal-to-proximal signaling is unknown. Three genes that act together during the establishment of tissue polarity, fat, four-jointed and dachsous, also influence the expression of Wingless in the proximal wing. fat is required cell autonomously by proximal wing cells to repress Wingless expression, and misexpression of Wingless contributes to proximal wing overgrowth in fat mutant discs. Four-jointed and Dachsous can influence Wingless expression and Fat localization non-autonomously, consistent with the suggestion that they influence signaling to Fat-expressing cells. dachs is identified as a gene that is genetically required downstream of fat, both for its effects on imaginal disc growth and for the expression of Wingless in the proximal wing. These observations provide important support for the emerging view that Four-jointed, Dachsous and Fat function in an intercellular signaling pathway, identify a normal role for these proteins in signaling interactions that regulate growth and patterning of the proximal wing, and identify Dachs as a candidate downstream effector of a Fat signaling pathway (Cho, 2004).
There is a progressive elaboration of patterning along the PD axis over the course of wing development. During the second larval instar, interactions among the Epidermal Growth Factor Receptor, Dpp and Wg signaling pathways divide the wing disc into a dorsal region, which will give rise to notum, and a ventral region, from which the wing will arise. An initial PD subdivision of the wing is then effected by signaling from the AP and DV compartment boundaries, which promotes the expression of two genes, scalloped and vestigial, that encode subunits of a heterodimeric transcription factor (Sd-Vg) in the center of the wing. This subdivides the wing into distal cells, which give rise to the wing blade, and surrounding cells, which give rise to proximal wing and wing hinge structures. The proximal wing is further subdivided into a series of molecularly distinct domains. Studies of Sd-Vg function in the wing led to the realization that the elaboration of this finer pattern depends in part upon signaling from the distal, Sd-Vg-expressing cells, to more proximal cells. Thus, mutation of vg leads to elimination, not only of the wing blade, where Vg is expressed, but also of more proximal tissue. Conversely, ectopic expression of Vg in the proximal wing reorganizes the patterning of surrounding cells (Cho, 2004 and references therein).
A key target of the distal signal is Wg, which during early third instar is expressed in a ring of cells that surround the SD-VG-expressing cells, and which later becomes expressed in a second, more proximal ring. Wg expression in the inner, distal ring within the proximal wing is regulated by an enhancer called spade-flag (spd-fg), after an allele of wg in which this enhancer is deleted (Neumann, 1996). Studies of this allele, together with ectopic expression experiments, have revealed that Wg is necessary and sufficient to promote growth of the proximal wing. Wg also plays a role in proximal wing patterning; it acts in a positive-feedback loop to maintain expression of Homothorax (Hth). The rotund (rn) gene has been identified as an additional target of distal signaling (Cho, 2004 and references therein).
This work identified Four-jointed (Fj), Dachsous (Ds), Fat and Dachs as proteins that influence signaling to proximal wing cells to regulate Wg and rn expression. Fj is a type II transmembrane protein, which is largely restricted to the Golgi. Null mutations in fj do not cause any obvious defects in the proximal wing. However, fj plays a role in the regulation of tissue polarity, yet acts redundantly with some other factor(s) in this process. Mutations in fat or ds can also influence tissue polarity. Although the molecular relationships among these proteins are not well understood, genetic studies suggest that fj and ds act via effects on fat, and both fj and ds can influence Fat localization in genetic mosaics (Cho, 2004 and references therein).
Interestingly, alleles of fj, ds and fat, as well as alleles of another gene, dachs, can result in similar defects in wing blade and leg growth. The similar requirements for these genes during both appendage growth and tissue polarity, together with the expression patterns of fj and ds in the developing wing, led to this investigation of their requirements for proximal wing development. All four genes influence the expression of Wg in the proximal wing, and genetic experiments suggest a pathway in which Fj and Ds act to modulate the activity of Fat, which then regulates transcription via a pathway that includes Dachs. These observations lend strong support to the hypothesis that Fj, Ds and Fat function as components of an intercellular signal transduction pathway, implicate Dachs as a key downstream component of this pathway, and identify a normal role for these genes in proximodistal patterning during Drosophila wing development (Cho, 2004).
The common feature of all of the manipulations of FJ and DS expression carried out in this study is that Wg expression, and by inference, Fat activity, can be altered when cells with different levels of Fj or Ds are juxtaposed. In the case of Fj, its normal expression pattern, and effects of mutant and ectopic expression clones are all consistent with the interpretation that juxtaposition of cells with different levels of Fj is associated with inhibition of Fat in the cells with less Fj and activation of Fat in the cells with more Fj. The influence of Ds, however, is more variable. Studies of tissue polarity in the eye suggest that Ds inhibits Fat activity in Ds-expressing cells, and/or promotes Fat activity in neighboring cells. The predominant effect of Ds during early wing development is consistent with this, but its effects in late discs are not. Studies of tissue polarity in the abdomen suggest that the Ds gradient might be interpreted differently by anterior versus posterior cells, and it is possible that a similar phenomena causes the effects of Ds to vary during wing development (Cho, 2004).
The influence of ds mutation on gene expression and growth in the wing is much weaker than that of fat. It has been suggested that Fj might influence Fat via effects on Ds, and fj mutant clones have been observed to influence Ds protein staining. The observations are consistent with the inference that both Ds and Fj can regulate Fat activity, but they do not directly address the question of whether Fj acts through Ds. They do, however, indicate that even the combined effects of Fj and Ds cannot account for FAT regulation, and, assuming that the strongest available alleles are null, other regulators of Fat activity must exist. It is presumably because of the counteracting influence of these other regulators that alterations in Fj and Ds expression have relatively weak effects. In addition, according to the hypothesis that Fat activity is influenced by relative rather than absolute levels of its regulators, the effects of Fj or Ds could be expected to vary depending upon their temporal and spatial profiles of expression, as well as on the precise shape and location of clones (Cho, 2004).
The observations imply the existence of at least two intracellular branches of the Fat signaling pathway. One branch involves the transcriptional repressor Grunge, influences tissue polarity, certain aspects of cell affinity, and fj expression, but does not influence growth or wg expression. An alternative branch does not require Grunge, but does require Dachs. Dachs is implicated as a downstream component of the Fat pathway, based on its cell autonomous influence on Fat-dependent processes, and by genetic epistasis. The determination that it encodes an unconventional myosin, and hence presumably a cytoplasmic protein, is consistent with this possibility. It also suggests that Dachs does not itself function as a transcription factor, and hence implies the existence of other components of this branch of the Fat pathway. This Grunge-independent branch influences Wg expression in the proximal wing and imaginal disc growth. However, further studies will be required to determine whether Dachs functions solely in Grunge-independent Fat signaling, or whether instead Dachs is required for all Fat signaling (Cho, 2004).
The observations that fj expression is regulated by Sd-Vg, and that fj is both necessary and sufficient to modulate the distal ring of Wg expression in the proximal wing, suggest that Fj influences the activity of a distal signal, which then acts to influence Fat activity. However, the relatively weak effects of fj indicate that other factors must also contribute to distal signaling, just as fj functions redundantly with other factors to influence tissue polarity. Since Ds expression is downregulated in a domain that is broader than the Vg expression domain, a direct influence of Vg on the Ds gradient is unlikely, and the essentially normal appearance of Wg expression in the proximal wing in fj ds double mutants implies that Ds is not a good candidate for the hypothetic factor Signal X. Rather, it is suggested that Ds acts in parallel to signaling from Vg-expressing cells to modulate Fat activity. This Vg-independent effect would account for the remnant of the distal ring that sometimes appears in vg null mutants. Importantly though, the observation that the phenotypes of hypomorphic dachs mutant clones on Wg expression are more severe than fj and ds suggests that the hypothesized additional factors also act via the Fat pathway. It is also noted that the limitation of Wg expression to the proximal wing even in fat mutant clones implies that Wg expression both requires Nubbin, and is actively repressed by distally-expressed genes (Cho, 2004).
The recovery of normal Wg expression by later stages in both fj and dachs mutant clones implies that the maintenance of Wg occurs by a distinct mechanism. Prior studies have identified a positive-feedback loop between Wg and Hth that is required to maintain their expression. It is suggested that once this feedback loop is initiated, Fat signaling is no longer required for Wg expression. Moreover, the recovery of normal levels of Wg at late stages suggests that this positive-feedback loop can amplify reduced levels of wg to near normal levels (Cho, 2004).
The distinct consequences of Vg expression and Fj expression in clones in the proximal wing suggest that another signal or signals, which are qualitatively distinct from the Fj-dependent signal, is also released from VG-expressing cells. When Vg is ectopically expressed, Wg is often induced in a ring of expression that completely encircles it. However, this is not the case for Fj-expressing clones. Both Vg- and Fj-expressing clones can activate rn and wg only within NUB-expressing cells, but Vg expression can result in non-autonomous expansion of the Nub domain, and this expansion presumably facilitates the expression of Wg by surrounding cells. Another striking difference between Vg- and Fj-expressing clones is that in the case of ectopic Fj, enhanced Wg expression is only in adjacent cells. By contrast, in the case of Vg, Wg expression initiates in neighboring cells, but often moves several cells away as the disc grows, resulting in a gap between Vg and Wg expression. This gap suggests that a repressor of Wg expression becomes expressed there, and recent studies have identified Defective proventriculus (Dve) as such a repressor (Cho, 2004).
In strong fat mutants, the wing discs become enlarged and have extra folds and outgrowths in the proximal wing. The disproportionate overgrowth of the proximal wing is due to upregulation of Wg in this region, as demonstrated by its suppression by wgspd-fg. At the same time, clones of cells mutant for fat overgrow in other imaginal cells, and fat wgspd-fg discs are still enlarged compared with wild-type discs. Thus, Fat appears to act both by regulating the expression of other signaling pathways (e.g. Wg), and via its own, novel growth pathway. The identification of additional components of this pathway will offer new approaches for investigating its profound influence on disc growth (Cho, 2004).
The dachs gene was first identified almost a century ago based on its requirements for appendage growth. This paper describes the phenotypes of strong dachs mutations, reports the cloning of the dachs gene, characterizes the localization of Dachs protein, and investigates the relationship between Dachs and the Fat pathway. Mutation of dachs reduces, but does not abolish, the growth of legs and wings. dachs encodes an unconventional myosin that preferentially localizes to the membrane of imaginal disc cells. dachs mutations suppress the effects of fat mutations on gene expression, cell affinity and growth in Imaginal discs. Dachs protein localization is influenced by Fat, Four-jointed and Dachsous, consistent with its genetic placement downstream of fat. However, dachs mutations have only mild tissue polarity phenotypes, and only partially suppress the tissue polarity defects of fat mutants. These results implicate Dachs as a crucial downstream component of a Fat signaling pathway that influences growth, affinity and gene expression during development (Mao, 2006).
The observation that a hypomorphic mutation of dachs could suppress the effects of fat mutations on wing growth and Wg expression in the proximal wing has led to the suggestion that dachs might act as a downstream component of a Fat signaling pathway. This study provides two types of evidence that confirm this suggestion. First, dachs is epistatic to fat for multiple phenotypes in multiple tissues, including gene expression, growth and cell affinity. Indeed, with the notable exception of the influence of fat on tissue polarity, all known fat mutant phenotypes are completely suppressed by mutation of dachs. Second, it was found that expression of regulators of Fat, Fj and Ds, or of Fat itself, influence the localization or stability of Dachs protein at the membrane, thus providing a molecular link from Fat to Dachs (Mao, 2006).
The predicted structure of Dachs is unique within the myosin superfamily, and places Dachs in a new class of unconventional myosins. It has most similarity to myosins V, VII, and X. This is intriguing, as a mammalian protocadherin, Cdh23, has been functionally linked to myosin VIIa during the development of sensory hair cells in the inner ear (Mao, 2006).
Within the myosin head region, the major conserved domains are all present, suggesting that Dachs functions as a motor protein. However, it is also possible that Dachs serves a structural or scaffolding role. For example, in the Hedgehog pathway, a kinesin-related protein, Costal2, is thought to function largely as a scaffold that brings together crucial kinases with their substrates (Mao, 2006).
The dGC2 mutation deletes part of the N terminal extension. As dGC2 mutants have relatively weak phenotypes, the N terminal extension might not be not essential for Dachs activity. Conversely, the severe phenotypes of alleles that truncate Dachs in the myosin head region imply that the myosin domain is essential. dGC13 in particular is predicted to eliminate almost all of the myosin head domain, and genetically it appears to act as a null allele (Mao, 2006).
Characterization of new dachs alleles has provided an opportunity to define more clearly the requirements for dachs. dachs is required for normal wing and leg growth, although some appendage growth is dachs independent. Importantly, the identification of dachs as a downstream component of a Fat signaling pathway that influences growth implies that the reduced growth in dachs mutants is reflective of a normal role for a Fat pathway in growth promotion. That is, while fat is a gene whose normal role can be thought of as to restrain growth, as mutant tissue overgrows, it is suggested that inhibition of Fat occurs during normal development, and that this inhibition contributes to normal appendage growth, as defined by the reduced growth of dachs mutants. Normal inhibition of Fat activity would presumably be effected by the two known regulators of Fat, Fj and Ds (Mao, 2006).
Whether available dachs mutations fully define the normal involvement of the Fat pathway in growth promotion is not yet clear. The possibility cannot be excluded that dachs is partially redundant with other proteins (e.g. other myosins), although this seems unlikely given the complete suppression of all non-polarity phenotypes of fat by dachs. It is also possible that dachs is required only for peak Fat signaling. This explanation is suggested by the observation that expression of the Fat target genes wg, Ser and fj is only partially or transiently lost in dachs mutants, yet the elevated or ectopic expression of these genes in fat mutants is completely eliminated by mutation of dachs (Mao, 2006).
The relatively mild tissue polarity phenotypes of dachs mutants, and the inability of dachs mutation to completely suppress the influence of fat on tissue polarity, contrast with the absolute dependence of fat gene expression, growth and affinity phenotypes on dachs. These observations suggest that there are two distinct Fat pathways. One, crucially dependent on Dachs, influences gene expression, growth and cell affinity, and another, partially independent of Dachs, influences tissue polarity. Studies of the atrophin protein Grunge also support the suggestion that there is a distinct Fat polarity pathway, as Grunge interacts with Fat and influences tissue polarity, but does not exhibit other phenotypes observed in fat mutants. Thus, Dachs might act redundantly with another protein in a polarity pathway, but non-redundantly in a pathway that influences gene expression. It should also be noted that effects of dachs on gene expression might contribute to the polarity phenotypes of dachs mutants. For example, fj is regulated by dachs, and fj has polarity phenotypes (Mao, 2006).
The asymmetric localization of Dachs observed in wild-type wings, and the influence of Fj and Ds on Dachs localization, have important implications for tissue polarity. (1) The asymmetric localization of Dachs is itself a form of polarity, and its detection in third instar imaginal discs emphasizes that these cells are polarized well before core polarity proteins such as Frizzled and Dishevelled become asymmetrically localization in pupal wings. A similar conclusion can be drawn from the recent observation that fat and ds influence the orientation of cell divisions in third instar discs. (2) The observations identify an ability to induce asymmetric protein localization as a mechanism through which the Fat pathway might influence tissue polarity. Dachs is one target, but the Fat polarity pathway might similarly involve asymmetric localization of other myosins, or of other types of proteins, to affect tissue polarity (Mao, 2006).
Mutation of fat is associated with elevated Dachs staining at the membrane, and overexpression of Fat decreases Dachs staining at the membrane. Although this negative effect of Fat on Dachs is subject to the caveat that only tagged overexpressed Dachs:V5 can be detected, this tagged protein rescues dachs mutants, and the effects of Fat on Dachs staining are consistent with their opposite phenotypes and the epistasis of dachs to fat. Manipulations of the expression of Fat regulators provide further evidence that Fat regulates Dachs levels at the membrane, and altogether these observations implicate Dachs as a crucial intracellular component of a Fat signaling pathway (Mao, 2006).
The concomitant elevation of Fat staining and loss of Dachs staining observed at the perimeter of Fj-expressing clones is consistent with the conclusion that Fat can antagonize the localization or stability of Dachs at the membrane. Because the elevation of Fat is limited to the periphery of Fj-expressing clones, it is hypothesized that it results from an influence of Fj on Fat-Ds interactions, rather than the expression of Fj per se. Tissue polarity studies have implied that Fj and Ds have opposite affects on Fat. Although it has not yet been determined whether Fj can directly modify Fat or Ds, the simplest explanation for the elevated Fat staining at the edge of Fj-expressing cells would be to propose that Fj modifies Ds to inhibit its interactions with Fat. In this case, Fat protein within Fj-expressing clones would be predicted to prefer to bind to Ds outside of the clone, and hence to accumulate at the clone perimeter, where it would then downregulate Dachs (Mao, 2006).
The interpretation of the elevated Dachs staining at the perimeter of Ds-expressing clones is more complex. Although Fat is elevated at the clone perimeter, the depletion of Fat from neighboring cells suggests that the elevated Fat staining largely reflects Fat outside of the clone, rather than in Ds-expressing cells. Given that dachs and fat influence transcriptional targets cell autonomously, and dachs acts genetically downstream of fat, the link between elevated Fat in one cell and elevated Dachs in a neighboring cells must be indirect. It might be that Ds can also influence Dachs localization, and does so in opposite fashion to Fat. According to this scenario, the elevated Fat staining in cells neighboring the clone would be reflective of high levels of Ds engaged by Fat at the clone perimeter, which would then recruit or stabilize Dachs at the membrane. However, mutation of ds did not result in any noticeable decrease of Dachs:V5 staining. Alternatively, it might be that Fat antagonizes the accumulation of Ds within the same cell. High Fat accumulation at the edge of one cell could then result in low Fat accumulation at the edge of its neighbor through this hypothesized downregulation of Ds. In this case, the elevated Dachs accumulation at the edge of Ds-expressing clones would be a consequence of low levels of Fat. This model would also imply that asymmetric localization of Fat could be propagated from cell to cell, which could have important consequences for Fat pathway regulation. However, there is as yet no evidence that Fat is asymmetrically localized at wild-type levels of Fj and Ds expression (Mao, 2006).
Recent studies in Drosophila of the protocadherins Dachsous and Fat suggest that they act as ligand and receptor, respectively, for an intercellular signaling pathway that influences tissue polarity, growth and gene expression, but the basis for signaling downstream of Fat has remained unclear. This study characterizes functional relationships among Drosophila tumor suppressors and identifies the kinases Discs overgrown and Warts as components of a Fat signaling pathway. fat, discs overgrown and warts regulate a common set of downstream genes in multiple tissues. Genetic experiments position the action of discs overgrown (dco) upstream of the Fat pathway component dachs, whereas warts acts downstream of dachs. Warts protein coprecipitates with Dachs, and Warts protein levels are influenced by fat, dachs and discs overgrown in vivo, consistent with its placement as a downstream component of the pathway. The tumor suppressors Merlin, expanded (ex), hippo, salvador (sav) and mob as tumor suppressor (mats) also share multiple Fat pathway phenotypes but regulate Warts activity independently. These results functionally link what had been four disparate groups of Drosophila tumor suppressors, establish a basic framework for Fat signaling from receptor to transcription factor and implicate Warts as an integrator of multiple growth control signals (Cho, 2006).
Since Dachs is required for loss of Wts protein in fat mutants, and Dachs encodes a large Myosin protein, a model was considered in which Dachs acts as a scaffold to link Wts to proteins that promote Wts proteolysis, analogous to the roles of Costal2 in Hedgehog signaling, or APC in Wnt signaling. This model predicts that Dachs should be able to bind to Wts. To evaluate this possibility, tagged forms of Dachs and Wts were coexpressed in cultured cells and assayed for coimmunoprecipitation. These experiments identified a specific and reproducible interaction between Dachs and Wts (Cho, 2006).
Recent studies have identified the transcriptional coactivator Yorkie (Yki) as a downstream component of the Hippo pathway and a substrate of Wts kinase activity. Phosphorylation of Yki by Wts inactivates Yki, and overexpression of Yki phenocopies wts mutation. The determination that the Fat tumor suppressor pathway acts through modulation of Wts thus predicts that Yki should also be involved in Fat signaling. When the influence of Yki overexpression was examined on Fat target genes, expression of Wg in the proximal wing, Ser in the proximal leg and fj in the wing and eye were each upregulated by Yki overexpression, consistent with the inference that Fat tumor suppressor pathway signaling acts through Yki (Cho, 2006).
In order to identify additional components of the Fat tumor suppressor pathway, advantage was taken of the observation that loss of fat in clones of cells is associated with an induction of Wingless (Wg) expression in cells just proximal to the normal ring of Wg expression in the proximal wing, reflective of its role in distal-to-proximal wing signaling. It was reasoned that this influence on Wg expression could be used to screen other Drosophila tumor suppressors for their potential to contribute to Fat signaling. Analysis of mutant clones in the proximal wing identified dco, ex, mats, sav, hpo and wts as candidate components of the Fat tumor suppressor pathway. As for fat, mutation of each of these genes is associated with induction of Wg expression specifically in the proximal wing, whereas Wg expression is not affected in more distal or more proximal wing cells. Although Wg expression often seems slightly elevated within its normal domain, the effect of these mutations is most obvious in the broadening of the Wg expression ring. The induction of Wg expression does not seem to be a nonspecific consequence of the altered growth or cell affinity associated with these mutations, since Wg expression is unaffected by expression of the growth-promoting microRNA gene bantam or by expression of genes that alter cell affinity in the proximal wing (Cho, 2006).
dco encodes D. melanogaster casein kinase I delta/epsilon. The overgrowth phenotype that gave the gene its name is observed in allelic combinations that include a hypomorphic allele, dco3, and it is this allele that is associated with induction of Wg. Null mutations of dco actually result in an 'opposite' phenotype: discs fail to grow, and clones of cells mutant for null alleles fail to proliferate. This is likely to reflect requirements for dco in multiple, distinct processes, as casein kinase I proteins phosphorylate many different substrates, and dco has been implicated in circadian rhythms, Wnt signaling and Hedgehog signaling (Cho, 2006).
Mer and ex encode two structurally related FERM domain-containing proteins. ex was first identified as a Drosophila tumor suppressor, whereas Drosophila Mer was first identified based on its structural similarity to human Merlin. Mutation of Mer alone causes only mild effects on imaginal disc growth, but Mer and ex are partially redundant, and double mutants show more severe overgrowth phenotypes than either single mutant. Consistent with this, elevation of Wg expression was observed in ex mutant clones (7/10 proximal wing clones induced Wg) and not in Mer mutant clones (0/8 clones), whereas Mer ex double mutant clones showed even more severe effects on Wg than ex single mutant clones. Because of the partial redundancy between Mer and ex, when possible, focus was placed for subsequent analysis on Mer ex double mutant clones (Cho, 2006).
Wts, Mats, Sav and Hpo interact biochemically, show similar overgrowth phenotypes and regulate common target genes. Mats, Sav and Hpo are all thought to act by regulating the phosphorylation state and thereby the activity of Wts. Mutation of any one of these genes is associated with upregulation of Wg in the proximal wing. The effects of sav (47/84 clones in the proximal wing induced Wg) and hpo (23/31 clones) were weaker than those of mats (19/19 clones) and wts (92/97 clones), but this might result from differences in perdurance or allele strength. Because sav, hpo and mats all act through Wts, focus for most of the subsequent analysis was placed on wts (Cho, 2006).
The observation that mutation of dco, Mer, ex, mats, sav, hpo or wts all share the distinctive upregulation of Wg expression in the proximal wing observed in fat mutants suggests that the functions of these genes are closely linked. To further investigate this, the effects of these tumor suppressors were characterized on other transcriptional targets of Fat signaling. Expression of the Notch ligand Ser is upregulated unevenly within fat mutant cells in the proximal region of the leg disc. A very similar upregulation occurred in dco3, Mer ex, and wts mutant clones. fj is a target of Fat signaling in both wing and eye imaginal discs, and fj expression was also upregulated in dco3, Mer ex, or wts mutant clones. The observation that these genes share multiple transcriptional targets in different Drosophila tissues implies that they act together in a common process (Cho, 2006).
The hypothesis that Fat pathway genes and Hippo pathway genes are linked predicts that not only should Fat target genes be regulated by Hippo pathway genes, but Hippo pathway target genes should also be regulated by Fat pathway genes. The cell cycle regulator CycE and the inhibitor of apoptosis Diap1 (encoded by thread) have been widely used as diagnostic downstream targets to assign genes to the Hippo pathway. Notably, then, clones of cells mutant for fat showed upregulation of both Diap1 and CycE protein expression. Genes whose expression is upregulated within fat mutant cells (such as wg, Ser and fj) have been shown previously to be induced along the borders of cells expressing either fj or dachsous (ds), and Diap1 is also upregulated around the borders of ds- or fj-expressing clones. That thread is affected by fat at a transcriptional level was confirmed by examining a thread-lacZ enhancer trap line. The regulation of Diap1 by the Hippo pathway is thought to be responsible for a characteristic eye phenotype in which an excess of interommatidial cells results from their failure to undergo apoptosis; an increase was also observed in interommatidial cells in fat mutant clones. Upregulation of both Diap1 and CycE is also observed in Mer ex double mutant clones. In dco3 mutant clones, consistent upregulation was detected only for Diap1, and CycE was upregulated only weakly and inconsistently. dco3 also has weaker effects on Wg and fj expression; the weaker effects of dco3 could result from its hypomorphic nature. ex has recently been characterized as another Hippo pathway target, and an ex-lacZ enhancer trap that is upregulated in wts or Mer ex mutant clones is also upregulated in fat or dco3 mutant clones. Analysis of ex transcription by in situ hybridization also indicated that ex is regulated by fat. Altogether, this analysis of Hippo pathway targets further supports the conclusion that the functions of the Fat pathway, the Hippo pathway and the tumor suppressors Mer, ex and dco are linked (Cho, 2006).
Genetic epistasis experiments provide a critical framework for evaluating the functional relationships among genes that act in a common pathway. The relationships was evaluated between each of the tumor suppressors linked to the Fat pathway and dachs, using both wing disc growth and proximal Wg expression as phenotypic assays. dachs is the only previously identified downstream component of the Fat tumor suppressor pathway. It acts oppositely to fat and is epistatic to fat in terms of both growth and gene expression phenotypes (Cho, 2006).
dachs is also epistatic to dco3 for overall wing disc growth and for proximal Wg expression. The epistasis of dachs to dco3 implies that the overgrowth phenotype of dco3 is specifically related to its influence on Fat signaling, as opposed to participation of dco in other pathways. By contrast to the epistasis of dachs to dco3, both wts and ex are epistatic to dachs for disc overgrowth phenotypes, and wts and Mer ex are epistatic to dachs in their influence on proximal Wg expression. Together, these epistasis experiments suggest that dco acts upstream of dachs, whereas Mer ex and wts act downstream of dachs (Cho, 2006).
Because wts and Mer ex have similar phenotypes, their epistatic relationship cannot be determined using loss-of-function alleles. However, overexpression of ex inhibits growth and promotes apoptosis, which suggests that ex overexpression affects ex gain-of-function. Clones of cells overexpressing ex are normally composed of only a few cells, and over time most are lost, but coexpression with the baculovirus apoptosis inhibitor p35 enabled recovery of ex-expressing clones. These ex- and p35-expressing clones were associated with repression of proximal Wg expression during early- to mid-third instar, as has been described for dachs2, consistent with ex overexpression acting as a gain-of-function allele in terms of its influence on Fat signaling. In epistasis experiments using overexpressed ex and mutation of wts, wts was epistatic; Wg was induced in the proximal wing. Additionally, when wts is mutant, coexpression with p35 was no longer needed to ensure the viability and growth of ex-expressing clones, indicating that wts is also epistatic to ex for growth and survival. Consistent with this conclusion, others have recently described phenotypic similarities between Mer ex and hpo pathway mutants and have reported that hpo is epistatic to Mer ex (Cho, 2006).
When Fat was overexpressed, a slight reduction was detected in Wg expression during early- to mid-third instar, suggesting that overexpression can result in a weak gain-of-function phenotype. Clones of cells overexpressing Fat but mutant for dco3 still showed reduced Wg levels, whereas clones of cells overexpressing Fat but mutant for warts showed increased Wg levels. Although experiments in which the epistatic mutation is not a null allele cannot be regarded as definitive, these results are consistent with the conclusion that wts acts downstream of fat and suggest that dco might act upstream of fat (Cho, 2006).
The epistasis results described above suggest an order of action for Fat tumor suppressor pathway genes in which dco acts upstream of fat, fat acts upstream of dachs, dachs acts upstream of Mer and ex, and Mer and ex act upstream of wts. However, the determination that one gene is epistatic to another does not prove that the epistatic gene is biochemically downstream, as it is also possible that they act in parallel but converge upon a common target. Thus, to better define the functional and hierarchical relationships among these genes, experiments were initiated to investigate the possibility that genetically upstream components influence the phosphorylation, stability or localization of genetically downstream (that is, epistatic) components. Focus in this study was placed on the most downstream of these components, Wts. As available antibodies did not specifically recognize Wts in imaginal discs, advantage was taken of the existence of functional, Myc-tagged Wts-expressing transgenes (Myc:Wts) to investigate potential influences of upstream Fat pathway genes on Wts protein. In wing imaginal discs, Myc:Wts staining outlines cells, suggesting that it is preferentially localized near the plasma membrane, and it was confirmed that expression of Myc:Wts under tub-Gal4 control can rescue wts mutation. Notably, mutation of fat results in a reduction of Myc:Wts staining. As Myc:Wts is expressed under the control of a heterologous promoter in these experiments, this must reflect a post-transcriptional influence on Wts protein. fat does not exert a general influence on the levels of Hippo pathway components; fat mutant clones had no detectable influence on the expression of hemagglutinin epitope-tagged Sav (HA:Sav) (Cho, 2006).
The decrease in Wts protein associated with mutation of fat contrasts with studies of the regulation of Wts activity by the Hippo pathway, which have identified changes in Wts activity due to changes in its phosphorylation state. To directly compare regulation of Wts by Fat with regulation of Wts by other upstream genes, Myc:Wts staining was examined in ex, sav and mats mutant clones. In each of these experiments, the levels and localization of Myc:Wts in mutant cells was indistinguishable from that in neighboring wild-type cells (Cho, 2006).
Since Myc:Wts appears preferentially localized near the plasma membrane, it was conceivable that the apparent decrease in staining reflected delocalization of Wts, rather than destabilization. To investigate this possibility, Wts levels were examined by protein blotting. Antisera against endogenous Wts recognized a band of the expected mobility in lysates of wing imaginal discs or cultured cells, and this band was enhanced when Wts was overexpressed. The intensity of this band was reproducibly diminished in fat or dco3 homozygous mutant animals but was not diminished in fat or dco3 heterozygotes or in ex mutants. Conversely, levels of Hpo, Sav, Mer or Mats were not noticeably affected by fat mutation (Cho, 2006).
The determination that Wts is affected by Fat, together with the genetic studies described above, place Wts within the Fat signaling pathway, as opposed to a parallel pathway that converges on common transcriptional targets. Indeed, given that even hypomorphic alleles of wts result in disc overgrowth, the evident reduction in Wts levels might suffice to explain the overgrowth of fat mutants. As a further test of this possibility, Wts levels were examined in fat dachs double mutants. As the influence of Fat on gene expression and growth is absolutely dependent upon Dachs, if Fat influences growth through modulation of Wts, its influence on Wts levels should be reversed by mutation of dachs. Examination of Myc:Wts staining in fat dachs clones and of Wts protein levels in fat dachs mutant discs confirmed this prediction (Cho, 2006).
Prior observations, including the influences of fat and ds on gene expression, and the ability of the Fat intracellular domain to rescue fat phenotypes, suggested that Fat functions as a signal-transducing receptor. By identifying kinases that act both upstream (Dco) and downstream (Wts) of the Fat effector Dachs and by linking Fat to the transcriptional coactivator Yki, these results have provided additional support for the conclusion that Fat functions as a component of a signaling pathway and have delineated core elements of this pathway from receptor to transcription factor. Fat activity is regulated, in ways yet to be defined, by Ds and Fj. The influences of Fat on gene expression, growth, and cell affinity, as well as on Wts stability, are completely dependent on Dachs, indicating that Dachs is a critical effector of Fat signaling. Since Dachs can associate with Wts or a Wts-containing complex, it is suggested that Dachs might act as a scaffold to assemble a Wts degradation complex. The observations that Fat, Ds and Fj modulate the subcellular localization of Dachs, that Wts is preferentially localized near the membrane and that Dachs accumulates at the membrane in the absence of Fat, suggest a simple model whereby Fat signaling regulates Wts stability by modulating the accumulation of Dachs at the membrane and thereby its access to Wts. The working model is that dco3 is defective in the phosphorylation of a substrate in the Fat pathway, but the recessive nature of dco3, the genetic epistasis experiments, and biochemical experiments argue that this substrate is not Wts, and further work is required to define the biochemical role of Dco in Fat signaling (Cho, 2006).
In addition to identifying core components of the Fat pathway, the results establish close functional links between the Fat pathway, the Hippo pathway and the FERM-domain tumor suppressors Mer and Ex. The common phenotypes observed among these tumor suppressors can be explained by their common ability to influence Wts. However, they seem to do this in distinct ways, acting in parallel pathways that converge on Wts rather than a single signal transduction pathway. The Fat pathway modulates levels of Wts, apparently by influencing Wts stability. By contrast, the Hippo pathway seems to regulate the activity of Wts by modulating its phosphorylation state. Thus, Wts seems to act as an integrator of distinct growth signals, which can be transmitted by both the Fat pathway and the Hippo pathway. It has been suggested that Mer and Ex also act through the Hippo pathway, although present experiments cannot exclude the possibility that Mer and Ex act in parallel to Hpo. Moreover, it should be noted that Mats might regulate Wts independently of Hpo and Sav and hence function within a distinct, parallel pathway. Although it is simplest to think of parallel pathways, there is also evidence for cross-talk. fj and ex are both components and targets of these pathways. Thus, they can be regarded as feedback targets within their respective pathways, but their regulation also constitutes a point of cross-talk between pathways. Another possible point of cross-talk is suggested by the observation that levels of Fat are elevated within Mer ex mutant clones. Although the potential for cross-talk complicates assessments of the relationships between tumor suppressors, the observations that fat, dco3 and dachs affect Warts protein levels in vivo, whereas ex, hippo, sav and mats do not, argues that there are at least two distinct pathways that converge on Warts. This conclusion is also consistent with the observations that ex, hippo, sav and mats can influence Wts phosphorylation in cultured cell assays, but Fat, Dachs and Dco do not (Cho, 2006).
Although the Fat and Hippo pathways converge on Wts, Hippo pathway mutants seem more severe. Thus, hpo, wts or mats mutant clones show a distinctive disorganization and outgrowth of epithelial tissues that is not observed in fat mutant clones, and they show a greater increase in interommatidial cells. This difference presumably accounts for the previous failure to recognize the tight functional link between Fat and Hippo signaling, and it can be explained by the finding that Wts levels are reduced but not completely absent in fat mutant cells. Thus, fat would be expected to resemble a hypomorphic allele of wts rather than a null allele, and consistent with this, a hypomorphic allele, wtsP2, results in strong overgrowth phenotypes. The effects of Yki overexpression on growth and target gene expression can be even stronger than those of fat or wts mutations, which suggests that Yki levels become limiting when upstream tumor suppressors are mutant (Cho, 2006).
fat encodes a protocadherin, which in the past has led to speculation that its influences on growth and cell affinity might result from Fat acting as a cell adhesion molecule. However, all of the effects of fat on growth and affinity require dachs, which is also required for the effects of fat on transcription. Additionally, targets of Fat signaling include genes that can influence growth and affinity; recent studies identified an influence of fat on E-cadherin expression, and as describe in this study, Fat influences CycE and Diap1 expression. Thus, one can account for the influence of fat on growth and affinity by its ability to regulate gene expression. fat interacts genetically with other signaling pathways, including EGFR and Wnt, and in some cells Fat signaling also influences the expression of ligands (such as Wg and Ser) for other signaling pathways. Regulation of these ligands contributes to fat overgrowth phenotypes, but since clonal analysis indicates that fat is autonomously required for growth control in most imaginal cells, the principal mechanism by which fat influences growth presumably involves the regulation of general targets (Cho, 2006).
Normal tissue growth and patterning depend on a relatively small number of highly conserved intercellular signaling pathways. The Fat pathway is essential for the normal regulation of growth and PCP in most or all of the external tissues of the fly and also participates in local cell fate decisions. In this regard, its importance to fly development can be considered comparable to that of other major signaling pathways. Although the biological roles and even the existence of a Fat pathway in mammals remain to be demonstrated, there is clear evidence that the mammalian Warts homologs Lats1 and Lats2 act as tumor suppressors and that a mammalian Yorkie homolog, YAP, can act as an oncogene. Moreover, other genes in the Drosophila Fat pathway have apparent structural homologs in mammals. Thus, it is likely that mammals also have a Fat tumor suppressor pathway that functions in growth control (Cho, 2006).
Organ growth is influenced by organ patterning, but the molecular mechanisms that link patterning to growth have remained unclear. The Dpp morphogen gradient in the Drosophila wing influences growth by modulating the activity of the Fat signaling pathway. Dpp signaling regulates the expression and localization of Fat pathway components, and Fat signaling through Dachs is required for the effect of the Dpp gradient on cell proliferation. Juxtaposition of cells that express different levels of the Fat pathway regulators four-jointed and dachsous stimulates expression of Fat/Hippo pathway target genes and cell proliferation, consistent with the hypothesis that the graded expression of these genes contributes to wing growth. Moreover, uniform expression of four-jointed and dachsous in the wing inhibits cell proliferation. These observations identify Fat as a signaling pathway that links the morphogen-mediated establishment of gradients of positional values across developing organs to the regulation of organ growth (Rogulja, 2008).
Studies of regeneration first led to models that proposed that growth could be influenced by gradients of positional values, with steep gradients promoting growth and shallow gradients suppressing growth. Experimental manipulations of Dpp pathway activity in the Drosophila wing supported this concept, but have left unanswered the question of how differences in the levels of Dpp pathway activity perceived by neighboring cells are actually linked to growth. This study has established that the Fat signaling pathway provides this link. Dpp signaling influences the Fat pathway; the expression of upstream Fat pathway regulators, the subcellular localization of Fat pathway components, and downstream transcriptional outputs of Fat signaling are all affected by Dpp signaling. The effects that Tkv and Brk expression have on the expression of Fat target genes parallels their effects on BrdU labeling and depend genetically on Fat signaling (Rogulja, 2008).
Dpp signaling impinges on Fat signaling upstream of Fat, as the expression of both of its known regulators, Fj and Ds, is regulated by Dpp signaling. Although the Fat signaling pathway was only recently discovered, and understanding of Fat signaling and its regulation remains incomplete, the inference that Fat signaling is normally influenced by the Dpp morphogen gradient is supported by the polarized localization of Dachs in wild-type wing discs. Near the D-V compartment boundary, the vector of Dachs polarization parallels the vector of the Dpp morphogen gradient, and the consequences of altered Dpp pathway activity confirm that the correlation between them is reflective of a functional link. The expression of Fj and Ds and the localization of Dachs are also polarized along the D-V axis. The implication that signaling downstream of the D-V compartment boundary thus also impinges on Fat signaling, and indeed may also influence growth through this pathway, is consistent with the observation that normal wing growth requires both A-P and D-V compartment boundary signals, and is further supported here by the observation that Notch activation affects both fj expression and Dachs localization (Rogulja, 2008).
The results argue that Fat signaling is influenced by the graded expression of its regulators: uniform expression of Fj and Ds can activate Fat signaling and thereby inhibit growth, whereas juxtaposition of cells expressing different levels of either Fj or Ds can inhibit Fat signaling and thereby promote growth. Here, a model is proposed to explain how Fat signaling can be modulated by Fj and Ds gradients. Although aspects of the model remain speculative, it provides an explanation for a number of observations that would otherwise appear puzzling, and serves as a useful framework for future studies (Rogulja, 2008).
Central to the model is the inference that the interaction between Ds and Fat activates Fat. This inference is well supported by the observations that mutation or downregulation of ds results in overgrowth and upregulation of Diap1, whereas uniform overexpression of Ds inhibits growth and Diap1 expression. A second key aspect of the model is that once activated by Ds, Fat locally transmits a signal to a complex at the membrane. An important corollary to this is that if Fat and Ds are not engaged around the entire circumference of a cell, then there could be a region where Fat is locally inactive. This is hypothetical, but the Fat-dependent polarization of Dachs implies that there can be regional differences in Fat activity within a cell. Local Fat signaling is then proposed to locally promote Warts stability and activity, and thereby locally antagonize Yki activity. Conversely, a local absence of Fat signaling could result in a local failure to phosphorylate Yki, which could then transit to the nucleus, where it would promote the expression of downstream target genes. Formally, this model treats Fat signaling like a contact inhibition pathway: if Fat is engaged by Ds around the entire circumference of a cell, then Fat is active everywhere and downstream gene expression is off; however, if Fat is not active on even one side of a cell, then Yki-dependent gene expression can be turned on and growth can be promoted (Rogulja, 2008).
In this model, graded expression of Fat regulators, like Fj and Ds, could modulate Fat signaling by polarizing Fat activity within a cell. In theoretical models of PCP, even shallow gradients of polarizing activity can be converted to strong polarity responses through positive-feedback mechanisms. How this might be achieved in Fat signaling is not yet clear, but the polarized localization of Dachs implies that, at some level, Fat activity is normally polarized in wild-type animals, even where the Fj and Ds expression gradients appear relatively shallow. Importantly, this polarization hypothesis provides a solution to the puzzle of how Ds could act as a ligand to activate Fat, yet inhibit Fat along the edges of Ds-expressing clones. In this model, Ds overexpression in clones polarizes Fat activity, possibly through its ability to relocalize Fat. This would allow a strong derepression of Yki on the side of the cell opposite to where Ds and Fat are actually bound, resulting in the induction of Yki:Scalloped target gene expression and promotion of cell proliferation. Propagation of this polarization, e.g., through the influence of Fat-Ds binding on Fat and Ds localization, might explain the spread of effects beyond immediately neighboring cells. Conversely, uniform expression of Ds would generate cells presenting a ligand that activates Fat and dampens the relative difference in expression levels between neighboring cells. Yki would thus remain sequestered around the entire cell circumference, consistent with the reduced growth and Diap1 expression observed. A dampening of gradients could also explain why the induction of Fat/Hippo target gene expression or BrdU labeling associated with clones expressing Ds, Fj, or TkvQ-D is biased toward cells outside of clones (Rogulja, 2008).
The hypothesis of Fat polarization and local signal transduction also suggests a solution to another puzzle. In terms of their effects on tissue polarity and Dachs localization, Fj and Ds always behave as though they have opposite effects on Fat. Conversely, in terms of their effects on cell proliferation and downstream gene expression, Fj and Ds behave as though they have identical effects on Fat. To explain this, it is proposed that Fj acts oppositely to Ds, by, for example, antagonizing Ds-Fat binding. The influence of Ds and Fj on polarity would be a function of the direction in which they polarize Fat activity, which, based on their effects on epitope-tagged protein Dachs:V5, is opposite. In contrast, their influence on downstream gene expression and growth would be a function of the degree to which they polarize Fat activity, which could be the same. In other words, their influence on polarity would be a function of the vector of their expression gradients, and their influence on growth would be a function of the slope. However, since Dachs:V5 generally appears to be strongly polarized, the actual interpretation of Fj and Ds gradients may involve feedback amplification and threshold responses rather than providing a continuous response proportional to the gradient slope (Rogulja, 2008).
The results have provided a molecular understanding of a how a gradient of positional values, established by the morphogen Dpp and reflected, at least in part, in the graded expression of Fj and Ds, can influence growth. However, it is clear that other mechanisms must also contribute to the regulation of wing growth. The relative contribution of Fat gradients to wing growth can be estimated by considering the size of the wing in dachs mutants, or when Fj and Ds are expressed ubiquitously, as, in either case, it would be expected that the derepression of Yki associated with normal Fat signaling gradients was abolished. In both cases, the wing is less than half its normal size. Fat signaling could thus be considered a major, but by no means the sole, mechanism for regulating wing growth. The determination that not all wing growth depends on the regulation of Fat activity fits with the observation that Dpp signaling promotes growth in at least two distinct ways, one dependent upon its gradient, and the other dependent upon its levels. Other models for wing growth, including a Vestigial-dependent recruitment of new cells into the wing, and an inhibition of Dpp-promoted wing growth by mechanical strain, have also been proposed. It is emphasized that these models are not incompatible with the conclusion that a Fat gradient influences growth. Rather, it is plausible, and even likely, that multiple mechanisms contribute to the appropriate regulation of wing growth. Indeed, it is expected that a critical challenge for the future will be to define not only the respective contributions of these or other mechanisms to growth control, but also to understand feedback and crosstalk processes that influence how these different mechanisms interact with each other (Rogulja, 2008).
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, appe6 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. appe6 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 appe6 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, appe1, appe3, and appe6 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. appe3 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. appe6 and appe1 contain nonsense mutations predicted to truncate the protein prior to or toward the end of the DHHC-CRD, respectively, and therefore appe6 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 appe6 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 appe6 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 appe6 clones did not affect PCP, whereas large clones only affected PCP in the regions of the wing where defects were observed in appe6 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. ftfd and ftG-rv are likely null alleles predicted to truncate Ft N-terminal to its transmembrane region. ftfd homozygotes and ftfd/ftG-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 ftfd; appe6 and ftfd/ftG-rv; appe6 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 appe6. Null dGC13 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 appe6 clones, dGC13 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 appe6 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 appe6 mutants and partly rescued the crossvein spacing and leg-joint defects of appe6. 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 appe6 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 appe6 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).
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, ftG (fat Gull) is a dominant antimorph. Bryant (1988) studied in some detail the phenotype of ft8 (previously known as fat floppy-disc, ftfd) 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: ftG-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. ftG-rv is a recessive lethal allele (Bryant, 1988) corresponding to the amorphic condition (Mahoney, 1991). Hemizygous ftG-rv [ftG-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 ftG-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 ftG-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 ftG-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 ftG-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 ftG-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 ftG-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, ftG-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 ftG-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 (ftG-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 ftG-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 ftG-rv, 1.7 in both ft18 and ft4), and higher cell (trichome) density (1.4 times in ftG-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 ftG-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 ftG-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. ftG-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 ftG-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 ftG-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 ftG-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 ftG-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 ftG-rv and less so for ft4). This at least accounts for 80% in ft18, 71% in ftG-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 (aprK568) a major reduction of the wing pouch. The double homozygote ft18 aPrK568 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, ftG and ftG-rv) shows that they encode truncated proteins containing different fractions of the cadherin region but lacking the EGF domains and more proximal domains. A ftG revertant ( ftG-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 ftG (Mahoney, 1991). Developmental genetic studies have been carried out in four alleles ( ft8, Bryant, 1988; ftG-rv, ft4 and ft18; Garioa, 2000). Of these alleles only the molecular nature of the ftG-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: ftG-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 ftG-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 Armover and/or Armunder. 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 Armover. 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 Armunder is always weaker and only dlgM52 enhances Armover to the same extent as zw3M11 (Greaves, 1999).
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).
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).
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 Dmcdc2E1-24 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, gigMe109, Dp110D945A) or an increase (EP(3)3622, fta13, 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 gigMe109, 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:
The autonomous effects on reduced clone size can result from the poor viability of mutant cells (l(3)Me10 or Dmcdc2E1-24), as shown in twin clonal analysis and cell death monitoring. The increased clone size of EP(3)3622, fta13 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, fta13 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).
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).
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),
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-rafGOF
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 rafGOF 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 rafDN 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
rafDN 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 rafDN 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).
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).
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).
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).
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 (vgON) and non-wing (vgOFF) 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 dso cells, Ft signaling activity is promoted both by the absence of Ds and by the Vg-dependent up-regulation of fj. However, in fto cells, Ft is absent and Vg down-regulates ds, rendering the cells equivalent to dso fto 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).
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).
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).
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 RasV12 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 RasV12 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 RasV12 was expressed. In this model, RasV12 drives NB differentiation in RasV12-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 (dsΔICD) or ft lacking its extracellular domain (ftΔECD) 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).
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date revised: 15 December 2011
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