four-jointed

DEVELOPMENTAL BIOLOGY

See the embryonic expression pattern of fj at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.

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).

Effects of Mutation or Deletion

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).

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date revised: 20 December 2004

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