four-jointed

Transcriptional Regulation

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expression; in the eye, Wingless promotes the expression of mirror (mrr), which encodes a negative regulator of fringe (unpublished observations of McNeill, Chasen, Papayannopoulos, Irvine, and Simon, cited by Papayannopoulos, 1998).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

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

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

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

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

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

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

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

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

Protein Interactions

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

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

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

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

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

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

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

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

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

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