Four-jointed

InteractiveFly: GeneBrief

Gene name - four-jointed

Synonyms -

Cytological map position - 55C1--3

Function - a type II transmembrane kinase

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

Symbol - fj

FlyBase ID: FBgn0000658

Genetic map position - 2-81.5

Classification - type 2 transmembrane protein

Cellular location - surface

NCBI link: Entrez Gene

fj orthologs: Biolitmine

Recent literature

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

DEVELOPMENTAL BIOLOGY

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

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

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

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

Given the role of fj in the establishment of polarity in the Drosophila eye and the significance of expression gradients for the establishment of planar polarity, fj expression was investigated in the developing wing using the enhancer detector fj^P1, which accurately mirrors the pattern of FJ mRNA expression in vivo. While fj expression in the third-instar wing imaginal disc is confined to the wing pouch the pattern of expression shown by the enhancer detector at 6 h after prepupal formation (APF) is already graded with lower levels present in proximal regions. The graded expression is retained at subsequent stages, as detected both with anti-beta-galactosidase antibodies and by LacZ activity staining. Although there is no antibody available against the fj protein, the fidelity of the fj-lacZ reporter suggests that fj transcripts (and by implication Fj protein) are present in a gradient in the developing pupal wing. In contrast to the pattern seen in the wing, the highest levels of fj enhancer trap expression present in the abdominal segments of young adults are apparently limited to two transverse bands of small cells located across the a3 and p1 regions of each segment. While these bands represent the highest levels of staining at this time, more heavily stained specimens also indicate that a lower level of uniform expression is also present superimposed on the a3/p1 pattern. While intriguing, it should be noted that planar polarity has already been established by the time abdomens can be stained and the pattern seen may not mirror fj expression present during polarity establishment (Zeidler, 2000).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

To test the relationships of these genes, two hypomorphic alleles of fj, fj⁴ and fj^N7 were used. The majority of legs of fj^N7 flies retain partial joints of a ball and socket morphology at the juncture between T2 and T3, while fj⁴ produces larger partial joints or complete joints at the T2/3 boundary. Introduction of one mutant copy of dachs, abl, ena, or Ser into these backgrounds significantly increases the severity of the hypomorphic phenotypes, while each of these genes by itself is wholly recessive in the leg. Thus, dachs, abl, ena, and Ser act as dominant enhancers of fj, suggesting these genes may be part of a common pathway or network (Buckles, 2001).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

The d^GC2 mutation deletes part of the N terminal extension. As d^GC2 mutants have relatively weak phenotypes, the N terminal extension might not be not essential for Dachs activity. Conversely, the severe phenotypes of alleles that truncate Dachs in the myosin head region imply that the myosin domain is essential. d^GC13 in particular is predicted to eliminate almost all of the myosin head domain, and genetically it appears to act as a null allele (Mao, 2006).

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

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

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

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

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

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

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

Delineation of a Fat tumor suppressor pathway

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Although the Fat and Hippo pathways converge on Wts, Hippo pathway mutants seem more severe. Thus, hpo, wts or mats mutant clones show a distinctive disorganization and outgrowth of epithelial tissues that is not observed in fat mutant clones, and they show a greater increase in interommatidial cells. This difference presumably accounts for the previous failure to recognize the tight functional link between Fat and Hippo signaling, and it can be explained by the finding that Wts levels are reduced but not completely absent in fat mutant cells. Thus, fat would be expected to resemble a hypomorphic allele of wts rather than a null allele, and consistent with this, a hypomorphic allele, wts^P2, results in strong overgrowth phenotypes. The effects of Yki overexpression on growth and target gene expression can be even stronger than those of fat or wts mutations, which suggests that Yki levels become limiting when upstream tumor suppressors are mutant (Cho, 2006).

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

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

Morphogen control of wing growth through the fat signaling pathway

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

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

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

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

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

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

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

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

Propagation of Dachsous-Fat planar cell polarity

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

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

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

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

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

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

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

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

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

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

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

EFFECTS OF MUTATION

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

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

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

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

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

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

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

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

Because fj-related phenotypes in the eye are associated with clonal boundaries, and because no suitable clonal markers that do not themselves obscure the polarity of mutant clonal tissue are available, clones lacking fj and marked with yellow were initially generated using the FLP/FRT technique and the amorphic fj^d1 allele. The effect of localized removal of fj activity in the wing was found to produce striking disruptions in the normal pattern of wing hair planar polarity and occasionally results in the loss of wing hairs within the presumptive mutant area. The occasional instances in which the yellow marker can be scored indicate that the phenotypes are associated with fj^d1 mutant tissue. When plotted together in a composite diagram showing the orientation of disrupted planar polarity associated with clones in many wings, an overall indication of the potential planar polarity phenotypes that can be induced is obtained. This pattern is similar to the phenotype observed in wings from individuals homozygous for hypomorphic fz alleles (Zeidler, 2000).

Loss-of-function mutant fj^d1 clones were generated in the wing marked by sha with which the precise boundary of the clone could be marked and any nonautonomous effects observed. While the sha¹ allele used has no significant nonautonomous effect, sha¹;fj^d1 double mutant clones sometimes display striking wing hair planar polarity phenotypes in the wild-type hairs on the proximal side of mutant patches. However, clones smaller than approximately 5x5 cells never give nonautonomous phenotypes. When the positions of clones that produce nonautonomous phenotypes are plotted, only clones present within certain areas are found to generate nonautonomous polarity phenotypes. All clones larger than approximately 5x5 cells produce nonautonomous phenotypes in a region distal to the posterior cross vein. Larger clones also produce strong nonautonomous phenotypes in a region between veins 3 and 4, and weaker phenotypes are observed in a large region in the center of the wing posterior to vein 4. The positions were plotted in which polarity phenotypes induced by unmarked clones were seen, and this was seen to extend throughout almost the entire wing blade, in marked contrast to the limited area in which nonautonomous phenotypes are seen (Zeidler, 2000).

While the severity and extent of the phenotypes generated depend on clone position and size, the inversion of wing hair polarity is consistently seen on the proximal side of clones, resulting in the hairs pointing from a region lacking fj expression (in the clone) to regions of higher level fj expression (in the proximal wild-type tissue). Thus both misexpression and loss-of-function experiments are consistent with wing hairs pointing towards high levels of Fj. It should also be noted that the swirls of hairs associated with the wild-type tissue proximal to sha¹;fj^d1 double clones have a tendency to point toward the posterior of the wing. This result appears to be consistent with the composite results obtained from unmarked fj^d1 single-mutant clones and may be a result of the higher levels of fj expressed at the posterior margin of the wing (Zeidler, 2000).

As in the wing, planar polarity in the Drosophila abdomen is also exhibited by the polarity of hairs and bristles present on the cuticle of much of each abdominal segment. Unlike in the wing, in which only negligible polarity defects are observed, fj homozygotes show hair polarity defects in the abdomen, albeit in a very limited region of each segment. In the a2 region, for about 50% of fj^d1 abdominal segments, the hairs show extensive 'swirls' in a restricted region close to the midline, with weaker phenotypes observed in almost all segments. In addition, two different classes of loss-of-function clones were generated. First sha¹;fj^d1 double mutant clones were generated to identify nonautonomous phenotypes. Interestingly, fj does not appear to generate planar polarity phenotypes in the a4, a5, or posterior regions of a3. However, clones positioned in a2 and the anterior of a3 do show clear nonautonomous hair inversions adjacent to the posterior margin of mutant tissue. While sha-marked clones mark the extent of mutant clones, this method precludes the analysis of planar polarity within mutant regions. Double-mutant clones lacking the body pigment marker yellow and fj^d1 were therefore induced such that yellow mutant bristles in the abdominal cuticle marked the position (but not the precise outline) of the clones. In contrast to the situation in the wing, autonomous phenotypes are found in essentially the same region as the nonautonomous effects described above. Clones in a3-a5 show no phenotype, while clones in anterior a3 result in major disruptions of hair polarity. Given the greater range of inversions apparent in yellow- versus sha-marked fj^d1 mutant clones, it is clear that the majority of inverted hairs are present within mutant tissue and that inversions are not restricted to adjacent wild-type cells (Zeidler, 2000).

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

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

In addition to the role of fj in planar polarity determination demonstrated here, mutations in the locus are associated with both the fusion of tarsal joints and the proximal-distal (PD) shortening of both leg and wing structures. During these investigations it became clear that many fj misexpression experiments generate PD phenotypes. The phenotypes associated with loss of fj were compared to those produced by uniform fj misexpression using the actin-Gal4/UAS-fj system. In comparison to legs from wild-type flies, legs from fj^d1 homozygotes are clearly shortened and display the characteristic fusion of the second and third tarsi. Defects in the legs of actin-Gal4/UAS-fj individuals are very similar and include PD axis shortening as well as tarsal fusion. While actin-Gal4-driven defects are striking, the ectopic fj expression driven by the patched-Gal4 line can produce much more severe phenotypes and can result in extreme PD axis shortening and loss of distal structures while the coxa, a proximal structure, appears largely unaffected (Zeidler, 2000).

Wild-type wings are longer than those lacking fj activity and those in which fj is uniformly expressed by the actin-Gal4/UAS-fj system. In order to better classify the phenotypes obtained, the lengths of different sections of the wing were measured and compared. This shows that while proximal regions of the wing are unaffected the region between the anterior and the posterior cross vein is significantly reduced in both the null mutant (fj^d1) and the enhancer detector P-element insertion (fj^P1) homozygotes as well as in the actin-Gal4/UAS-fj-misexpressing individuals. While the effect on more proximal structures is more variable the overall wing lengths are significantly reduced in all cases. Thus actin-Gal4/UAS-fj produces PD defects both qualitatively and quantitatively similar to those produced from the total removal of fj in a homozygous amorphic situation. In addition to the previously characterized role of fj in leg outgrowth fj mutant individuals were also observed to have shorter and 'dumpier' abdominal regions than wild type. In order to quantitate this observation the width of the region of abdominal segments between the a1/a2 and the a5/a6 boundaries was quantitated. While wild-type abdomens are an average of 261 mm at the dorsal midline, fj^P1 homozygous abdomens are 221 mm in width. Other fj mutants show similar effects. Thus it is clear that the contraction of PD growth observed in the leg and wing also occurs in the abdomen (Zeidler, 2000).

A number of additional observations also indicate a link between fj and the patterning of wing veins. Beginning at about 18 h APF down-regulation of fj enhancer trap expression is seen in longitudinal stripes in the wing, which ultimately resolve to a pattern that is thought to represent the future veins. In addition to this, wings homozygous mutant for fj often contain ectopic wing vein material as do fj^d1 mutant clones in which vein material forms within or adjacent to the clone. Furthermore misexpression by actin-Gal4/UAS-fj also produces wing vein phenotypes including loss of the posterior cross vein and displacement of vein 4 at the junction with the wing margin as well as the deletion of the anterior cross vein in the region of fj misexpression in patched-Gal4/UAS-fj wings. In some sha¹;fj^d1 double-mutant clones, veins are often diverted or duplicated around mutant regions so that the vein material is situated in wild-type tissue immediately abutting the clonal boundary and in some cases veins 'fade' in mutant tissue. These results imply that fj acts as a regulator of wing vein formation and suggest that in normal development fj might function to ensure the precise positioning of the veins (Zeidler, 2000).

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

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

Previous studies have demonstrated that four-jointed (fj), which encodes a type II transmembrane/secreted protein, is expressed in a graded fashion from the equator toward the poles of the eye imaginal disc. Fj has been implicated in the regulation of ommatidial polarity based on the observation that reversals of ommatidial polarity occur along the polar border of fj mutant clones. However, fj mutant flies and the interior of fj clones only rarely display polarity defects. This has suggested that much of Fj action during ommatidial polarity formation may be redundant with other signals. The role of Fj was investigated further by asking whether Fj might regulate Ds or Ft function. To determine whether Fj acts predominantly in one member of the R3/R4 precursor pair, strong loss-of-function fj mutant (fj^N7) clones were generated, and R3/R4 mosaic ommatidia were examined. The loss of Fj function from the polar precursor cell, which expresses lower levels of Fj, had little effect on ommatidial polarity (6% reversals in 70 cases). In contrast, loss of Fj function from only the equatorial precursor cell led to polarity reversals in 71% of the 52 cases. These results indicate that Fj is mainly required in the equatorial R3/R4 precursor cell during the determination of ommatidial polarity. However, when only the equatorial cell lacks functional Fj, the Fj produced by the polar cell frequently is able to reverse the pattern of R3/R4 specification. These data suggest that the Fj gradient may play a role in directing R3/R4 specification by providing more Fj activity to the equatorial precursor cell (Yang, 2002).

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

The first prediction was tested by examining fj clones generated in ds^UA071 flies. Since the lack of a recognizable equator in homozygous ds mutant eyes made it impossible to designate normal versus reversed polarity ommatidia in fj^N7 clones, the fj R3/R4 mosaic ommatidia were analyzed by measuring the frequency with which the fj^-/- precursor cell became R4. When fj mosaic ommatidia were assayed in ds mutant flies, the fj^-/- precursor cell was equally likely to become R3 or R4 (54% as R4, 46% as R3). In contrast, the fj^-/- cell became R4 in 84% of cases when fj mosaic clones were generated in a ds⁺ background. These data show that differences in Fj function can only specify the pattern of R3/R4 specification when Ds is present. The ability of differences in Ds levels to influence R3/R4 specification in the absence of Fj function was examined by generating ds^UA071 clones in either wild-type or homozygous fj^N7 flies. When ds mutant clones were induced in wild-type background, the ds^-/- cell has 68% chance of becoming an R3. Interestingly, when ds clones were induced in fj animals, this frequency was increased to 85%, showing that the lack of Fj not only fails to block the action of Ds but instead enhances the ability of Ds differences to dictate the pattern of R3/R4 specification. These results support a model in which Fj acts upstream of Ds, perhaps as a regulator of Ds function (Yang, 2002).

Since fj and ds are expressed in opposing gradients in the eye, one possible mechanism by which Fj could regulate Ds function would be to inhibit ds transcription. This possibility was investigated by examining ds-lacZ expression in clones of cells that either lack Fj function or overexpress Fj. The lack of detectable effect on ds-lacZ expression in these experiments indicates that Fj does not act by antagonizing ds expression (Yang, 2002).

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

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

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

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

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

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

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

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

Four-jointed and abdominal compartmentalization

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

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

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

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

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

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

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

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

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

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

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

Now consider clones at the front of P; these clones would be expected to reverse the P cells behind them, and they do. However, they might also be expected to reverse the cells in the A compartment in front of them, but they do not. Perhaps polarity effects crossing over from P to A are blocked. There is some evidence that normally, Hh induces cells in posterior A (say, the a6 region) to sequester and/or destroy ambient Fj. This would create a local sink for Fj at the point farthest from its source and help build the gradient of Fj. Overexpression of fj at the back of A might make enough protein to overcome this sink, thus creating an ectopic peak of Fj extending both anteriorly into A and posteriorly across the A/P boundary into the P compartment, with consequent polarity reversal in both compartments. By contrast, overexpression of Fj in P (or by just one row of cells at the back of A) might not generate sufficient Fj across the boundary to overwhelm the sequestering activity of A cells, so only cells in P would see an ectopic peak and be repolarized (Casal, 2002).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

But this new model raises yet another challenge: consider the pleura, which is formed by a sheet of cells spanning several segments, all of which secrete hairs that point posteriorly. If, for example, the localization of Fz in each cell were controlled by the graded activity of Ft, then these Ft gradients would need to be precisely coextensive with the compartments. Otherwise, some cells would read gradients with the wrong sign and make hairs that point in the wrong direction. This could be most critical at the boundaries between the A and P compartments, where the gradient landscape of Ft should be forming peaks or troughs and hence might be relatively flat. This challenge could be resolved if, in a later and/or independent process, cell polarity were locally coordinated: there is some evidence for this. For example, clones of cells that lack or overexpress Fz can cause local reversals in hair polarities that propagate a few cell diameters beyond the clone borders (Casal, 2002).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

EVOLUTIONARY HOMOLOGS

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

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

REFERENCES

Search PubMed for articles about Drosophila four-jointed

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

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Biological Overview

date revised: 25 April 2019