Table of contents

Wingless targets in the eye disc

The eye imaginal disc displays dorsal-ventral (D-V) and anterior-posterior polarity prior to the onset of differentiation, which initiates where the D-V midline intersects the posterior margin. As the wave of differentiation progresses anteriorly, additional asymmetry develops as ommatidial clusters rotate coordinately in opposite directions in the dorsal and ventral halves of the disc; this forms the equator, a line of mirror-image symmetry that coincides with the D-V midline of the disc. The currently unanswered question of how D-V pattern is established and how it relates to ommatidial rotation was addressed by assaying the expression of various asymmetric markers under conditions that lead to ectopic differentiation, such as removal of patched or wingless function. D-V patterning is found to develop gradually. wingless plays an important role in setting up this pattern. To determine if positional information associated with equatorial formation is present along the D-V axis of the disc ahead of the MF, expression of an equatorial marker (WR122, a lacZ insertion in an unknown locus) was studied in various genetic conditions that lead to ectopic neuronal differentiation. This expression is dependent on the activity of the gene frizzled, which is required for proper ommatidial rotation. Induction of patched mutant clones activates the Hedgehog pathway and leads to precocious neuronal differentiation.Ectopic ommatidia that arise in clones show that the potential to express WR122-lacZ is restricted to neurons located near the D-V midline, regardless of their position along the A-P axis of the disc. This suggests that the information necessary to restrict WR122 expression exists ahead of the MF (Heberlein, 1998).

The expression of WR122 was examined under conditions that reduce Wingless activity. A temperature sensitve wg allele was used. A reduction in Wg function during the late larval stages promotes precocious differentiation in the eye disc. This differentiation starts from the dorsal (and to a lesser degree the ventral) margin and proceeds inward, roughly perpendicular to the direction of progression of the normal differentiation front. Expression of WR122 is unrestricted among ectopic ommatidia that differentiate as a consequence of reduced Wg function. The normal expression domain of the marker is broadened toward the lateral margins. It is concluded that the expression of WR122 is inhibited by Wg in ommatidia located near the disc's margin, which restricts expression to the equatorial region. Ectopic expression of Wg is sufficient to repress WR122 expression in the more central portions of the retinal epithelium. Thus Wg functions to restrict the expression of the WR122 marker. wingless is necessary and sufficient to induce dorsal expression of the gene mirror prior to the start of differentiation and also to restrict the expression of the WR122 marker to differentiating photoreceptors near the equator. Manipulations in wingless expression shift the D-V axis of the disc as evidenced by changes in the expression domains of asymmetric markers, the position of the site of initiation and the equator, and the pattern of epithelial growth. Thus, Wg appears to coordinately regulate multiple events related to D-V patterning in the developing retina (Heberlein, 1998).

The Hedgehog (Hh) and Epidermal growth factor receptor (Egfr) signaling pathways play critical roles in pattern formation and cell proliferation in invertebrates and vertebrates. In this study, a direct link between these two pathways is demonstrated in Drosophila. Hh and Egfr signaling are each required for the formation of a specific region of the head of the adult fruitfly. hh and vein (vn), which encodes a ligand of the Drosophila Egfr, are expressed in adjacent domains within the imaginal primordium of this region. Using loss- and gain-of-function approaches, it has been demonstrated that Hh activates vn expression. Hh activation of vn is mediated through the gene cubitus interruptus (ci) and this activation requires the C-terminal region of the Ci protein. wingless (wg) represses vn expression, thereby limiting the domain of EGFR signaling (Amin, 1999).

Polarity of the Drosophila compound eye is established at the level of repeating multicellular units (known as ommatidia), which are organized into a precise hexagonal array. The adult eye is composed of ~800 ommatidia, each of which forms one facet. Sections through the eye reveal that each ommatidium contains eight photoreceptor cells in a stereotypic trapezoidal arrangement that has two mirror-symmetric forms: a dorsal form present above the dorsoventral (DV) midline, and a ventral form below. An axis of mirror-image symmetry runs along the DV midline and is known as the equator. By analogy to the terrestrial equator, the extreme dorsal and ventral points of the eye are referred to as the poles. Differentiation of ommatidia begins during the third instar larval stage when a furrow moves from posterior to anterior over the epithelium of the eye imaginal disc. Each ommatidial unit is born as a bilaterally symmetrical cluster of photoreceptor precursors, that is polarized on its anteroposterior axis. The clusters then become polarized on the DV (or equatorial-polar) axis, by the process of proto-ommatidium rotation via two 45° steps away from the DV midline, forming the equator. It has been suggested that the direction of this rotation is a consequence of a gradient of positional information emanating from either the midline or the polar regions of the disc (Zeidler, 1999a and references).

A number of recent studies have shed light on some of the mechanisms involved in the positioning of the equator on the DV midline of the eye imaginal disc. It is now clear that a critical step is the activation of Notch activity in a line of cells along the midline, and that this localized activation of Notch is a consequence of the restricted expression of the fringe (fng) gene product in the ventral half of the disc and the homeodomain transcription factor Mirror (Mirr) in the dorsal half of the disc. Furthermore, an important role for Wingless (Wg) in polarity determination on the DV axis has been demonstrated. Wg is a secreted protein (and the founder member of the Wnt family of morphogens) that is expressed at the poles of the eye disc. Wg has been shown to act as an activator of mirr expression; increasing the levels of Wg expression in the eye disc shifts mirr expression and the position of the equator ventrally, whereas reduction of wg function shifts mirr expression dorsally. Additionally, it has been shown convincingly that a gradient of Wg signaling across the DV axis of the eye disc regulates ommatidial polarity such that the lowest point of Wg signaling coincides with the equator (Zeidler, 1999a and references).

The JAK/STAT pathway is central to the establishment of planar polarity during Drosophila eye development. A localized source of the pathway ligand, Unpaired/Outstretched, present at the midline of the developing eye, is capable of activating the JAK/STAT pathway over long distances. A gradient of JAK/STAT activity across the DV axis of the eye regulates ommatidial polarity via an unidentified second signal. Additionally, localized Unpaired influences the position of the equator via repression of mirror (Zeidler, 1999a).

The data points to a model in which Upd and Wg first act to define the equator via restriction of mirr expression to the dorsal hemisphere and localized activation of Notch along the DV midline. Definition of the equator is known to occur early in development, while the disc is still small, and divides the disc into two hemispheres separated by a straight boundary that will form the future equator. Such boundaries evidently serve as a source of a second signal that can polarize ommatidia, since fng loss of function clones that induce ectopic regions of activated Notch result in changes in ommatidial polarity. Subsequently in development, it is surmised that gradients of JAK/STAT and Wg-pathway activity across the DV axis of the eye disc are responsible for setting up a gradient(s) of one or more second signals (most likely detected by the receptor Frizzled) that can determine ommatidial polarity. These signals might be responsible for maintaining longer range polarization of ommatidia away from the equator and the localized Notch-dependent polarizing signal (Zeidler, 1999a and references).

From these characteristics, the following can be deduced: the nonautonomy of the phenotype produced by removal of the autonomously acting pathway component JAK, and its dependence on clone size and shape, suggests that JAK/STAT affects ommatidial polarity via a secreted downstream signal (which subsequently will be referred to as a second signal, most likely detected by Frizzled). The direction of the nonautonomy (only in a polar direction) and the strict DV nature of the polarity inversions indicates that this second signal must be graded in its activity along the DV axis, with a change in direction of the gradient at the equator. The direction of this gradient would then be the instructive cue to which ommatidia respond when rotating to establish their mature polarity (Zeidler, 1999a).

The simplest model would be that there is a single second signal secreted from the equator, which is downstream of mirr/fng/Notch, and that Wg and Upd/JAK/STAT feed into this pathway upstream of Notch. This is consistent with the roles of Wg and Upd as regulators of mirr expression and, thus, in positioning the endogenous equator. However, it is not consistent with the observed ommatidial polarity inversions produced in the eye field both dorsally and ventrally by Wg-pathway and JAK/STAT-pathway LOF and GOF clones. These phenotypes indicate that second-signal concentration is dependent on Wg pathway and JAK/STAT pathway activity across the whole of the eye field, and thus the second signal cannot be only secreted from the DV midline as a consequence of localized Notch activation. It is conceivable that Notch is activated on the polar boundary of JAK/STAT LOF clones, but in this context the only known mechanism of Notch activation is via mirr/fng interactions, and this possibility has been ruled out (Zeidler, 1999a).

Instead, the data points to a model in which Upd and Wg first act to define the equator via restriction of mirr expression to the dorsal hemisphere and localize activation of Notch along the DV midline. Definition of the equator is known to occur early in development, while the disc is still small, and divides the disc into two hemispheres separated by a straight boundary that will form the future equator. Such boundaries evidently serve as a source of a second signal that can polarize ommatidia, becausefng LOF clones that induce ectopic regions of activated Notch result in changes in ommatidial polarity (Zeidler, 1999a).

Subsequently in development, it is surmised that gradients of JAK/STAT and Wg-pathway activity across the DV axis of the eye disc are responsible for setting up a gradient(s) of one or more second signals that can determine ommatidial polarity. These signals might be responsible for maintaining longer range polarization of ommatidia away from the equator and the localized Notch-dependent polarizing signal. A number of observations provide a great deal of support for such a model. (1) It is consistent with the known timing of the events involved. The requirement for fng function has been shown to lie between late first instar and mid second instar, which coincides with the first appearance of high levels of Upd expression at the optic stalk. However, the ommatidia are not formed (and thus do not respond to the polarity signal) until the start of the third instar, a stage when localized Upd expression still persists. Furthermore, extracellular Upd protein can be seen in a concentration gradient many cell diameters from the optic stalk at the early third instar stage, consistent with Upd being at least partly responsible for setting up the long-range gradient of JAK/STAT activity across the DV axis of the eye disc that is revealed by the stat92E-lacZ reporter. (2) This model does not require that a single source of second signal secreted by a narrow band of cells at the equator should be capable of determining ommatidial polarity across the whole of the DV axis of the disc during the third instar stage of development. Instead, the band of activated Notch at the equator would serve to draw a straight line between the fields of dorsally and ventrally polarized ommatidia, and need only secrete a localized source of second signal to polarize ommatidia in this region. Further from the equator, the opposing gradients of Upd and Wg signaling would provide a robust mechanism for maintenance of correct ommatidial polarity across the DV axis. Conversely, without the mirr/fng/Notch mechanism to draw a straight line, it would be impossible to imagine how Upd at the posterior margin and Wg at the poles alone could provide the perfectly straight equator that is ultimately formed. (3) The phenotypes that are observed are consistent with multiple competing mechanisms responsible for determining ommatidial polarity. When inversions of ommatidial polarity are induced by generating hop clones or ectopically expressing Upd, straight equators are not produced, such that two cleanly abutting fields of dorsal and ventral ommatidia are produced. Instead, there is usually some confusion of ommatidial identities as if they might be receiving conflicting signals. Additionally, when upd activity is removed from the optic stalk, an equator still forms (albeit at the ventral edge of the disc), but some ommatidia dorsal to the equator still adopt a ventral fate as if the determination of ommatidial polarity is less robust in the absence of Upd (Zeidler, 1999a).

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, 1999b).

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, 1999b).

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, 1999b).

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, 1999b).

The dorsal head capsule, which lies between the compound eyes, contains three morphologically distinct domains. The medial domain includes the ocelli and their associated bristles, which lie on the triangular ocellar cuticle. The mediolateral region contains the frons cuticle, which consists of a series of closely spaced parallel ridges. The lateral region is occupied by the orbital cuticle, which contains a stereotypical pattern of bristles. The head capsule forms primarily from the two eye-antennal imaginal discs. Each half of the dorsal head derives from a primordium in the disc immediately adjacent to the anlage of the compound eye. During the pupal stage, the two discs fuse at what will form the midline of the dorsal head capsule (Amin, 1999).

wingless is broadly expressed throughout the early eye-antennal disc, where it confers a default state of head cuticle. Later, wg expression becomes restricted to the primordia of the orbital cuticle and ptilinium, and to a portion of the antennal anlage. Just as hh expression is medially adjacent to that of vn on the adult head capsule, wg expression abuts vn in the frons both laterally and anteriorly. Loss of Wg signaling causes the deletion of both the frons and orbital cuticles. To determine whether Wg participates in vn regulation, a temperature-sensitive allele was used to eliminate Wg function during second instar development. In contrast to Hh, Wg negatively regulates vn. Loss of Wg activity during this time window expands the domain of vn expression in the dorsal head primordium and induces ectopic vn expression in other regions of the eye-antennal disc (Amin, 1999).

The Drosophila eye is patterned by a dorsal-ventral organizing center mechanistically similar to those in the fly wing and the vertebrate limb bud. Here it is shown how this organizing center in the eye is initiated -- the first event in retinal patterning. Early in development, the eye primordium is divided into dorsal and ventral compartments. The dorsally expressed homeodomain Iroquois genes are true selector genes for the dorsal compartment; their expression is regulated by Hedgehog and Wingless. The organizing center is then induced at the interface between the Iroquois-expressing and non-expressing cells at the eye midline. It was previously thought that the eye develops by a mechanism distinct from that operating in other imaginal discs, but this work establishes the importance of lineage compartments in the eye and thus supports their global role as fundamental units of patterning (Cavodeassi, 1999).

The formation of the DV midline has been postulated to appear de novo in an initially homogeneous eye field via a mechanism that involves gradients of secreted signaling molecules, like Wg, expressed at the disc margin. Accordingly, the position and shape of the eye midline are defined at the point of lowest concentration of a dorsal (Wg) and a ventral (unidentified) signal and prior to the subdivision of the disc into dorsal and ventral expression domains. One way these signals might enable the DV midline to become the organizer is by inducing the expression of IRO-C genes in the dorsal cells. According to the results presented in this study, affinity differences between dorsal (IRO-C +) cells and ventral (IRO-C -) cells may be the main mechanism responsible for maintaining the straight DV midline. To investigate how the two models are reconcilable, the expression of IRO-C and wg was examined at first/early second instar stages and their putative regulatory relationships were studied by clonal analysis. At late first/early second instar, IRO-C expression is already restricted to the dorsal half of the disc. A groove marking the limit of IRO-C expression, resembles that found along the presumptive DV boundary in the early third instar eye discs, as previously described. Differences in affinity between dorsal and ventral cells probably induce this groove because dorsal IRO-C clones are also transiently surrounded by a fold. At early second instar wg is expressed in the presumptive dorsal region of the eye disc. Later this expression evolves to dorsal and ventral anterior marginal domains. Expression of IRO-C was assayed in cells lacking dishevelled activity and therefore unable to transduce Wg signaling. Early and late induced dsh clones autonomously lack ara/caup expression, indicating that Wg is required continuously for IRO-C expression. This expression is normally downregulated in cells posterior to the morphogenetic furrow but it is maintained in dorsal-posterior clones of shaggy mutant cells, where the Wg pathway is constitutively active. However, activation of IRO-C in ventral sgg M1-1 clones is seen only occasionally in a subset of the mutant cells. Hence, it is concluded that Wg signaling is necessary but not sufficient to activate IRO-C expression (Cavodeassi, 1999).

Early generalized ectopic expression of hh dorsalizes the eye, severely reducing its size. Similar effects have been reported for early misexpression of wg. Together, these observations and the previous data support a model in which both Wg and Hh signaling organize DV patterning by directing IRO-C expression. However, Wg and Hh do not meet the complete requirement for the postulated gradient model: (1) their expression is already asymmetric in the early disc; (2) ubiquitous and high expression of Wg or Hh should prevent the formation of the straight DV boundary, but this is not the case (Cavodeassi, 1999).

Retinal differentiation is associated with the passage of the morphogenetic furrow, which normally begins at the intersection of the DV midline with the posterior margin. The site of furrow initiation is widely assumed to be specified at the lowest point of concentration of Wg activity. IRO-C expression borders can non-autonomously recruit mutant and wild-type cells to form an eye provided they are located close to the disc margin. Thus, IRO-C may induce retinal differentiation through the local repression of wg at the disc margin, causing a sink of the Wg gradient. Therefore the expression of wg was examined in relation to IRO-C borders. At late second/early third instar, wg is expressed around the anterior dorsal and ventral disc margins. wg expression is not impeded within marginal IRO-C mutant clones. Thus, it is concluded that an IRO-C expression border is sufficient to promote furrow initiation, even in the presence of wg (Cavodeassi, 1999).

The Drosophila eye disc is a sac of single layer epithelium with two opposing sides: the peripodial membrane (PM) and the disc proper (DP). Retinal morphogenesis is organized by Notch signaling at the dorsoventral (DV) boundary in the DP. Functions of the PM in coordinating growth and patterning of the DP are unknown. The secreted proteins Hedgehog, Wingless, and Decapentaplegic are expressed in the PM. From there they control DP expression of the Notch ligands Delta and Serrate. Peripodial clones expressing Hedgehog induce Serrate in the DP while loss of peripodial Hedgehog disrupts disc growth. Furthermore, PM cells extend cellular processes to the DP. Therefore, peripodial signaling is critical for eye pattern formation and may be mediated by peripodial processes (Cho, 2000).

Restricted localization of Hh-, Wg-, and Dpp-LacZ+ expressing cells along the DV axis in L1 discs suggests that these signals might act upstream of N. To test this idea, Hh activity was removed using a temperature-sensitive allele; Wg was ectopically expressed using hs-wg, or Dpp activity was removed by using a heteroallelic combination of the two dpp alleles, and then the expression patterns of the N ligands Dl and Ser were visualized in the eye discs. In L2 wild-type discs, Dl is preferentially expressed in the dorsal domain, while Ser is enriched along the DV midline of the DP. Both Dl and Ser are also present in the PM at a low level. In hhts2 mutants shifted to restrictive temperature during the early L1 stage, both Dl and Ser are uniformly expressed in dorsal and ventral domains. Ubiquitous Wg overexpression causes variable defects in Dl pattern such as significant reduction in the dorsal domain except near the margin or mislocalization to the ventral domain. Wg overexpression also causes mislocalization of Ser to the dorsal DP. dppe12/dppd14 mutant discs showed similar disruption of the DV-specific Dl and Ser pattern, indicating the necessity of Hh, Wg, and Dpp in DV patterning (Cho, 2000).

The complex interplay of Hh, Wg, and Dpp signaling has been studied for initiation and progression of the morphogenetic furrow. This study has examined much earlier stages of eye development to determine whether these same molecules organize DV patterning prior to retinal differentiation; it has been demonstrated that: (1) Hh, Wg, and Dpp display distinct DV expression patterns in the PM in early discs; (2) their signals are essential for domain-specific expression of Dl and Ser in the DP, and (3) signaling from the PM to DP is important for patterning in the DP. These findings provide a novel view of how eye discs are patterned, a model suggesting Hh, Wg, and Dpp signal from the PM to the DP by means of cellular processes (Cho, 2000).

Soon after the embryo hatches, wg- and dpp-LacZ+ cells appear in the dorsal and ventral domains of the disc, respectively. This suggests that the eye disc is already subdivided into dorsal and ventral fates. Consistent with this data, analyses of genetic mosaics have indicated that the eye disc consists of dorsal and ventral compartments of different cell lineages and of different cell affinities. Subsequent to the initial appearance of wg- and dpp-LacZ+ cells, these two types of cells are juxtaposed in the DV midline of PM and seem to be mutually exclusive in later stages. Such an antagonistic interaction between Wg and Dpp is a common theme that has emerged from studies in limb disc patterning and may play a crucial role in defining domains in the PM (Cho, 2000).

In addition to DV subdivisions, the dorsal domain of L1 discs appears to be further divided into anterior-posterior subdomains. This is based on the expression of Wg in the anterior but not in the posterior dorsal domain, while Dpp is expressed in the opposite pattern. The anterior and posterior subdomains may correspond to the anlage for the head and the dorsal eye, respectively. It has been shown that Wg expressed in the vertex and gena primordia is important for head capsule formation, while Dpp is antagonistic to this process. Interestingly, many new types of wg-LacZ+ PM cells appear during the L2 stage and occupy either DV midline or anterior dorsal domain. Perhaps some of these wg-LacZ+ PM cells may play important roles in specifying head fate of the anterior dorsal domain (Cho, 2000).

The PM is an important source of inductive signals to control cell fates within the DP. According to the presented model, Hh acts differentially to localize Wg- and Dpp-expressing cells to the dorsal and ventral domains of the PM, respectively, in the L1 disc. Establishment of DV domains in the PM governs subsequent signaling from the PM to the DP for controlling the DV specificity and the level of Dl/Ser expression. This idea is supported by observations that ectopic Hh expression in the PM cells can induce Ser expression in the DP, consistent with spatiotemporal correlation of Hh and Ser expression pattern in the L1 and L2 discs (Cho, 2000).

Although this study has focused on signaling from the PM to the DP, the signaling may be bidirectional. It is conceivable that the extension of peripodial processes may depend on specific signaling cues provided from the DP. Such bidirectional signaling may be essential to coordinate DV boundary formation and disc growth in both layers. Whether the signaling molecules that are transferred from the PM cells are Hh/Wg/Dpp themselves and/or other molecules remains unanswered. Interestingly, Patched (Ptc), the receptor for Hh that is known to be upregulated transcriptionally by Hh signaling, is expressed in the DP but is more abundant in the PM. This suggests that Hh signaling may occur laterally and vertically, within the PM layer as well as between the two layers (Cho, 2000).

Inductive signaling between two cell layers is an important mechanism of morphogenesis in vertebrate development. For instance, BMP4 signaling between optic vesicle and surface ectoderm is important for lens induction in vertebrates. Wnt signaling between the ectoderm and the mesoderm is also crucial for proper dorsoventral limb patterning. First shown to occur during Drosophila leg disc regeneration and now in the eye, peripodial signaling to the DP may be analogous to such inductive signaling in vertebrates. This study illustrates a novel mechanism of interepithelial signaling between PM and DP layers and its importance in eye disc patterning. Significantly, ablation or genetic disruption of the PM also affects development of the DP, providing additional evidence for peripodial signaling. Precise localization of receptors and downstream components for Hh, Wg, and Dpp in early eye discs will help in understanding how these signals are transmitted between the PM and the DP (Cho, 2000).

The bHLH transcription factor Atonal is sufficient for specification of one of the three subsets of olfactory sense organs on the Drosophila antenna. Misexpression of Atonal in all sensory precursors in the antennal disc results in their conversion to coeloconic sensilla. The mechanism by which specific sense organ fate is triggered remains unclear. The homeodomain transcription factor Cut, which acts in the choice of chordotonal-external sense organ does not play a role in olfactory sense organ development. The expression of atonal in specific domains of the antennal disc is regulated by an interplay of the patterning genes, Hedgehog and Wingless, and Drosophila epidermal growth factor receptor pathway (Jhaveri, 2000).

Pattern formation in the epidermis is regulated by a hierarchy of genes; the patterning genes -- engrailed, hh, dpp and wg -- specify co-ordinates of the disc and are expected to influence expression of prepatterning genes. Lz is a putative prepatterning gene in the antennal disc and has been shown to regulate expression of amos; genes regulating ato in the antenna are as yet unclear. The olfactory sense organs are located in a distinct pattern across the antenna, thus requiring co-ordinated control of the different proneural genes (Jhaveri, 2000).

During Drosophila eye development, Hh and Dpp are required to initiate photoreceptors at the furrow while Wg inhibits differentiation at the lateral margins. Wg appears to act by antagonizing signaling through the Egfr pathway. In contrast, Hh may directly regulate ato expression, its diffusion ahead of the morphogenetic furrow turns on Ato, while higher levels behind the furrow lead to its downregulation. There is however evidence that Hh can also influence Egfr signaling since Ci has been shown to activate Mapk through the Egfr ligand Vein (Jhaveri, 2000 and reference therein).

Loss-of-function experiments have shown that Hh function is required for ato expression; misexpression analysis has demonstrated that low Hh levels turn on Ato, while higher levels suppress it. However the normal expression pattern of Hh in the antennal disc makes it unlikely that it could directly act to induce Ato in all domains. Ectopic expression of wg in the antennal disc has been shown to lead to induction of ato. Hh appears to act non-autonomously to induce Ato in neighboring cells; UAS-hh transgene produces the secreted form of Hh protein. The data suggests a dosage sensitivity in the regulation of ato by Hh. High levels of Hh produced within cells of the clone suppress ato expression, while low levels resulting from diffusion of protein outside the clone induce it. It is thus proposed that both Hh and Wg together pattern ato expression domains in the disc. The diffusible nature of Hh could allow its action at a long range to induce expression of ato as well as wg. Since Wg is also a secreted molecule, and can regulate ato through the Egfr cascade, it could serve to extend the range of Hh effect across the disc (Jhaveri, 2000).

In the developing eye, wingless activity represses proneural gene expression (and thus interommatidial bristle formation) and positions the morphogenetic furrow by blocking its initiation in the dorsal and ventral regions of the presumptive eye. Evidence is provided that wingless mediates both effects, at least in part, through repression of the basic helix-loop-helix protein Daughterless. daughterless is required for high proneural gene expression and furrow progression. Ectopic expression of wingless blocks Daughterless expression in the proneural clusters. This repression, and that of furrow progression, can be mimicked by an activated form of armadillo and blocked by a dominant negative form of pangolin/TCF. Placing daughterless under the control of a heterologous promoter blocks the ability of ectopic wingless to inhibit bristle formation and furrow progression. hedgehog and decapentapleigic can not rescue the wingless furrow progression block, indicating that wingless acts downstream of these genes. In contrast, Atonal and Scute, which are thought to heterodimerize with Daughterless to promote furrow progression and bristle formation, respectively, can block ectopic wingless action. These results are summarized in a model where daughterless is a major, but probably not the only, target of wingless action in the eye (Cadigan, 2002).

Overexpression of Wingless using P[sev-wg] results in flies lack interommatidial bristles, due to Wg repression of proneural gene expression. To utilize this phenotype as a starting point to identify genes that interact with wg, P[sev-wgts] flies were created that express a temperature-sensitive form of Wg. At 25°C, these animals have the normal (600/eye) number of bristles. At 16°C, where the Wgts protein is almost fully active, less than 50 bristles remain. At 17.6°C, approximately 150-200 bristles form. This temperature was chosen to generate a sensitized background with which to screen for dominant modifiers (Cadigan, 2002).

Focus was placed on three enhancers of the P[sev-wgts] bristle phenotype. All three reduce the number of bristles to between 10-50/eye. These modifiers form one lethal complementation group, which was meiotically mapped to an area between 30-32 on the cytological map. Complementation with deficiencies narrowed the region to 31B-32A, a location that includes the da gene. Four lines of evidence demonstrate that these enhancers are alleles of da: (1) they fail to complement lethal alleles of da and are rescued by a P[da+] rescue construct; (2) null alleles of da dominantly enhance the P[sev-wgts] phenotype; (3) clones of two modifiers were negative for Da antibody staining; (4) identical effects on proneural gene expression, bristle formation and MF progression were observed in clones of the modifiers and known alleles of da (Cadigan, 2002).

Mechanosensory bristles are four-cell external sensory organs that are derived from single sensory organ precursors (SOPs). In the wing and eye, SOP specification requires the proneural genes ac and sc, which encode bHLH transcription factors. These genes are first expressed in groups of cells known as proneural clusters. As one cell reaches the threshold of Ac/Sc expression necessary to trigger SOP formation, it represses proneural gene expression in the other cells in the cluster. This occurs through a process referred to as lateral inhibition, involving Notch signaling (Cadigan, 2002).

The Ac and Sc proteins are thought to promote SOP formation by acting with Da, another bHLH protein. da alleles dominantly suppress the ectopic bristle phenotypes caused by the misexpression of sc and lethal of scute (lsc), a gene that mimics ac/sc. Da can bind to Ac or Sc, and the heterodimers can bind to specific DNA sequences known as E boxes. In cultured cells, Da and Ac or Sc synergistically activate reporter genes with promoters containing E boxes, including the proximal promoter of the ac gene. Unlike Ac and Sc, all cells examined express some Da, though there is significant modulation of levels in some embryonic tissues and the eye. Because most of the spatial information is manifested in Ac and Sc, Da is thought of as a proneural gene co-factor (Cadigan, 2002).

To further examine the relationship between Da and Ac/Sc and bristle formation, clones of da were examined in the eye and wing. While large clones in the eye result in a total lack of eye development because of a block in MF progression, small clones differentiate eye tissue that completely lack interommatidial bristles. Ac expression is reduced at 3 hours after pupae formation (h APF) in da clones. This reduction of Ac protein and sc mRNA expression is also seen in the presumptive wing margin. Thus, da is required for normal proneural gene expression, most likely at the level of autoactivation, preventing the high expression levels needed for SOP specification (Cadigan, 2002).

At 3h APF, every cell in the basal portion of the eye (where Ac is expressed) expresses Da. However, groups of two to three cells have a much higher level of expression. Double staining with Ac indicates that these are the proneural clusters. In P[sev-wg] eyes, where Ac expression is greatly reduced, Da expression is also significantly lower. In P[GMR-Gal4], P[UAS-wg] (GMR/wg) eyes, ectopic wg is expressed at a much higher level than P[sev-wg]. GMR/wg eyes have almost no detectable Da or Ac expression (Cadigan, 2002).

Does the ability of ectopic wg to repress Da and Ac expression reflect a normal role for wg in bristle inhibition? Normal adult eyes lack interommatidial bristles at the periphery of the eye. An inverse correlation between Wg expression and that of Ac was found at the edge of early pupal eyes. However, clones removing wg activity are identical to wild type, with no extra bristles at the eye's periphery. Clones of arm, in contrast, almost always cause bristles to form right up to the edge of the adult eye. Since loss of Arm activity is normally associated with a block in Wg signaling, this result suggests that endogenous Wg signaling may repress bristle formation at the periphery (Cadigan, 2002).

The lack of ectopic bristles in wg clones could be explained by the fact that wg clones often act non cell-autonomously due to Wg diffusing in from surrounding wg+ tissue. However, temperature shifts (from 12 hours before pupation to 12 h APF) with wgts animals results in only occasional ectopic bristles. Clones of Df(2L)RF exhibit ectopic bristles one third of the time. This deficiency has reported breakpoints of 27F2-4-28A3. While it may delete up to 30 annotated genes besides wg (27F3), these include three other Wnt genes; Dwnt4 (27E7-27F1) Dwnt6 (27F3-5) and Dwnt10 (27F5-6). The removal of Dwnt4 was confirmed by in situ hybridization of Df(2L)RF homozygous embryos. Thus it is possible that one or more of these Wnts acts through arm to repress bristle formation at the edge of the eye (Cadigan, 2002).

Expression of wg at high levels behind the furrow (via the GMR promoter) results in a dramatically reduced eye completely lacking bristles. The reduced eye size is not due to a lack of MF progression. GMR/wg eyes have a large degree of apoptosis during pupal development that is partially responsible for the reduction in eye size. Coexpression of wg with a dominant negative form of TCF (pangolin-FlyBase), the transcription factor that mediates many Wg transcriptional effects, suppresses the size reduction of the GMR/wg eye and bristle inhibition. Ac and Da levels are also greatly elevated compared to GMR/wg/lacZ controls. These results, plus the requirement for arm shown for P[sev-wg] indicate a canonical Wnt pathway mediating these effects (Cadigan, 2002).

Since Wg signaling represses both Da and Ac expression, and Ac expression depends on da activity, it is possible that Wg represses Ac through inhibition of Da. One piece of evidence in support of this is that Da levels are only modestly affected in animals lacking ac and sc. Because lack of ac/sc does not cause lack of Da, the simplest model is that Ac is repressed by Wg signaling due to Da inhibiton (Cadigan, 2002).

To further test this hypothesis, attempts were made to rescue the Wg-induced bristleless phenotype by coexpression with either Da or Sc. If the simple model were correct, placing da under a heterologous (i.e., GMR) promoter would restore bristles to GMR/wg eyes, but expressing sc in the same way would not. If GMR/wg represses both da and sc directly (by direct, it is meant without influence of the other gene), then neither da or sc heterologous expression would restore bristles (Cadigan, 2002).

Surprisingly, the results do not follow either of the above models. Both da and sc coexpression rescue the bristleless phenotype of GMR/wg eyes. Not every bHLH protein can rescue the bristles; GMR/wg/ato eyes are still completely bristleless. The GMR/wg/da eyes have a significant but modest increase in Ac levels while GMR/wg/sc eyes show a similar degree of increase of Ac and Da expression. These results suggest a more complicated situation, though caution is needed when interpreting overexpression studies (Cadigan, 2002).

In addition to its role in SOP specification, da is also known to be required for the initiation and progression of the MF. Da is expressed at higher levels in the MF than elsewhere in the eye imaginal disc. It is thought to form heterodimers with the bHLH protein Atonal to specify R8 differentiation, which then promotes MF progression (Cadigan, 2002).

wg is known to be required for the proper orientiation of the MF. Removal of wg causes ectopic furrow initiation from the dorsal and ventral borders of the eye disc. In addition, ectopically expressed wg can block MF initiation and progression. Having established that Wg blocks bristle formation through (at least in large part) Da repression prompted an investigation of the possibility of a similar connection in MF initiation (Cadigan, 2002).

Misexpression of wg using a Dpp-Gal4 driver, which is active at the posterior edge of the eye disc, causes a complete block in MF initiation. Co-expression with the dominant negative TCF construct significantly rescues the block. Expression of an activated form of arm also blocks MF initiation, though not quite to the same extent as wg. As with bristle inhibition, wg appears to block MF initiation through a canonical Wnt pathway (Cadigan, 2002).

The strategy to test an involvement of da in wg-mediated MF inhibition is similar to that employed in the investigation of bristles. Coexpression of da with wg with Dpp-Gal4 always restores some MF progression. da also causes a dramatic increase in MF progression in Dpp/armact eyes. Similar to what was found for sc with bristle formation, expression of ato with wg results in a significant rescue of MF progression. The degree of rescue is less than that observed with da. Expression of sc with wg also gives a modest rescue of the MF, with 7 of the 12 Dpp/wg/sc eyes examined showing some MF progression. This result is surprising, since sc has no known physiological role in regulating the MF and highlights the potential pitfalls of overexpression studies (Cadigan, 2002).

The genes dpp and hh have also been implicated in MF initiation and progression. Coexpression of dpp with wg does not result in any rescue. Dpp-Gal4 driving P[UAS-wg] and P[UAS-hh] never results in MF rescue from the posterior edge. However, the majority of the eyes had at least one ectopic furrow that initiated from the anterior portion of the eye. These furrows apparently had different initiation times, since some were quite small, some had progressed to several rows of concentric photoreceptors and some had progressed to fill most of the eye disc. At least in regard to the initiation of the endogenous furrow at the posterior end of the eye, the results suggest that Wg acts at the level of da/ato, rather than hh or dpp (Cadigan, 2002).

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).

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 armH8.6 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 Wingless protein plays an important part in regional specification of imaginal structures in Drosophila, including defining the region of the eye-antennal disc that will become retina. Wingless signaling establishes the border between the retina and adjacent head structures by inhibiting the expression of the eye specification genes eyes absent, sine oculis and dachshund. Ectopic Wingless signaling leads to the repression of these genes and the loss of eyes, whereas loss of Wingless signaling has the opposite effects. Wingless expression in the anterior of wild-type discs is complementary to that of these eye specification genes. Contrary to previous reports, it has been found that under conditions of excess Wingless signaling, eye tissue is transformed not only into head cuticle but also into a variety of inappropriate structures (Baonza, 2002).

In order to analyse the effect of ectopic activation of the Wingless pathway during the development of the eye-antennal imaginal disc, clones either mutant for the negative regulator of Wingless signaling, Axin, or expressing an activated form of Armadillo (Arm*) were induced. The loss of eye identity caused by the ectopic activation of Wingless, suggests a possible function for Wingless in the regulation of the eye selector genes. The top of the genetic hierarchy involved in eye specification appears to be the Pax6 homolog, Eyeless. In the third instar eye disc the expression of Eyeless is restricted to the region anterior to the furrow and, despite the Wingless-induced inhibition of eye development, the expression of Eyeless in this region is not affected by axin- clones. This lack of an effect anterior to the furrow, despite the overgrowth and abnormal Distal-less expression in the same region, implies that misregulation of Eyeless is not the primary cause of the transformations caused by ectopic Wingless activity (Baonza, 2002).

Downstream of Eyeless (although feedback relationships makes the epistatic relationship complex) are other transcription factors required for eye specification, including Eyes absent, Sine oculis and Dachshund. A phenotype similar to axin- clones of excess proliferation and consequent overgrowth is caused by loss of Eyes absent and Sine oculis. Moreover, as in axin- clones, clones mutant for sine oculis ectopically express Eyeless in the region posterior to the furrow. The similar mutant phenotypes shown by the loss of function of these genes and the ectopic activation of Wingless signaling make them good candidates to be regulated by the Wingless pathway (Baonza, 2002).

The expression patterns of Eyes absent, Sine oculis and Dachshund in axin- and/or arm* mutant clones were examined in third instar eye discs. At this stage, Dachshund is expressed at high levels on either side of the morphogenetic furrow, whereas Eyes absent and Sine oculis are expressed in all the cells of the eye primordium. In order to produce large patches of mutant tissue, the Minute technique was used. In axin- M+ clones the expression of Eyes absent in front of the furrow is always autonomously eliminated. This effect is not only seen in large clones that touch the eye margin but also in small internal clones. Identical results were obtained with Sine oculis and Dachshund: their expression was autonomously lost from anterior axin- M+ clones. Consistent with these results, in arm*-expressing clones Eyes absent, Dachshund and sine oculis (detected with a lacZ reporter construct) are similarly autonomously eliminated. It is therefore concluded that Wingless signaling represses the expression of the eye selector genes eyes absent, dachshund and sine oculis anterior to the morphogenetic furrow. Posterior to the furrow, however, some clones express high levels of Eyes absent, and Dachshund. This effect is always associated with overgrowth, and this expression is restricted to only some cells in these clones (Baonza, 2002).

The conclusion that Wingless signaling negatively regulates the expression of Eyes absent, Dachshund and Sine oculis anterior to the furrow leads to the prediction that in normal development, domains of high Wingless activity in the anterior region of the eye disc will be associated with low expression of these genes. Previous work indicates that their expression is broadly non-overlapping, but to analyse this precisely, discs were double-labelled to detect the expression of Wingless and Eyes absent or Sine oculis throughout the third instar larval stage. The expression of these eye specification genes is precisely complementary to that of Wingless in the anterior lateral margins of the eye throughout the third instar. This is consistent with a role for Wingless signaling in initiating the borders between eye and other head structures. Note that in posterior lateral regions slight overlap is observed between the expression of Wingless and these genes; this is presumably analagous to the expression of eye specification genes seen in some posterior axin- clones, and confirms that in posterior regions of the eye disc, Wingless signaling is not incompatible with the expression of these genes (Baonza, 2002).

These results indicate that Wingless regulates the final size of the eye field of cells by controlling the expression of eyes absent, sine oculis and dachshund. The expression pattern of these genes in the anterior eye margin is complementary to the expression of Wingless throughout the third instar, indicating that in anterior regions, high activity of Wingless signaling corresponds to absence of these gene products. Moreover, ectopic activation of Wingless signaling represses their expression anterior to the furrow (where they act to specify the eye field) throughout eye development. Finally, the loss of Wingless signaling causes ectopic expression of Eyes absent and Dachshund (Baonza, 2002).

It is proposed that the initial expression of Eyes absent, Sine oculis and Dachshund is negatively regulated by Wingless signaling in the eye disc, and that this regulation initiates the border between the eye field and adjacent head cuticle. Attempts were made to define whether Wingless represses the eye specification genes independently or whether eyes absent is the primary target but the data confirms earlier reports of the complexity of the regulatory relationships between eyes absent, sine oculis and dachshund. The observation that Eyes absent is able partially to restore the expression of the other two genes but cannot rescue the overgrowth and differentiation phenotype of axin- clones has two possible explanations. Either Wingless represses eye development through at least one additional gene, or high level Wingless signaling blocks eye development later in the developmental program -- e.g., it is known to inhibit morphogenetic furrow initiation, even after its earlier effects are rescued by eyes absent expression (Baonza, 2002).

Wingless signaling is required to distinguish wing pouch cells from notum cells. A similar function occurs for Wingless during eye disc development. Wg defines the border between retina and adjacent head. However, in addition to dorsal head cuticle, axin mutant cells can transform the eye cells into other tissues. For example, axin- clones sometimes express Distal-less, a gene not expressed in the third instar eye but specific to the leg, wing and antennal discs. This fits with reports that ectopic expression of Wingless during the development of other imaginal discs can induce transdetermination -- the change of cell identity from one fate to another. The plasticity of mammalian cells during development is a hotly debated issue that has important implications for the potential utility of stem cells. It may be that, as in other fields, Drosophila genetics can shed some light on the mechanisms of developmental plasticity and how such mechanisms are regulated (Baonza, 2002).

Odd-skipped genes are targeted by wingless and act specify the signaling center that triggers retinogenesis in Drosophila

Although many of the factors responsible for conferring identity to the eye field in Drosophila have been identified, much less is known about how the expression of the retinal 'trigger', the signaling molecule Hedgehog, is controlled. This study shows that the co-expression of the conserved odd-skipped family genes at the posterior margin of the eye field is required to activate hedgehog expression and thereby the onset of retinogenesis. The fly Wnt1 homologue wingless represses the odd-skipped genes drm and odd along the anterior margin and, in this manner, spatially restricts the extent of retinal differentiation within the eye field (Bras-Pereira, 2006).

The eye disc is a flat epithelial sac. By early third larval stage (L3), columnar cells in the bottom (disc proper: Dp) layer are separated by a crease from the surrounding rim of cuboidal margin cells. Margin cells continue seamlessly into the upper (peripodial; Pe) layer of squamous cells. The Dp will differentiate into the eye, while the margin and Pe will form the head capsule. In addition, the posterior margin produces retinal-inducing signals (Bras-Pereira, 2006).

By examining gene reporters it was found that the zinc-finger gene odd is expressed restricted to the posterior margin and Pe of L3 eye discs. Since the odd family members drumstick (drm), brother of odd with entrails limited (bowl) and sister of odd and bowl (sob) are similarly expressed in leg discs, they were examined in eye discs. In L2, before retinogenesis has started, odd and drm are transcribed in the posterior Pe-margin, and this continues within the posterior margin after MF initiation. bowl is transcribed in all eye disc Pe-margin cells of L2 discs, but retracts anteriorly along the margins and Pe after the MF passes. In addition, bowl is expressed weakly in the Dp anterior to the furrow. sob expression in L2 and L3 is mostly seen along the lateral disc margins. Therefore drm, odd and bowl are co-expressed at the posterior margin prior to retinal differentiation initiation (Bras-Pereira, 2006).

Odd family genes regulate diverse embryonic processes, as well as imaginal leg segmentation. In embryos, the product of the gene lines binds to Bowl and represses its activity, while Drm relieves this repression in drm-expressing cells. Since drm/odd/bowl expression coincides along the posterior margin around the time retinal induction is triggered, it was asked whether they controlled this triggering. First, bowl function was removed in marked cell clones induced in L1. bowl- clones spanning the margin, but not those in the DP, cause either a delay in, or the inhibition of, retinal initiation and the autonomous loss of hh-Z expression. Correspondingly, there is a reduction in expression of the hh-target patched (ptc). These effects on hh and ptc are not due to the loss of margin cells, since drm is still expressed in the bowl- cells. The requirement of Bowl for hh expression is margin specific, since other hh-expressing domains within the disc are not affected by the loss of bowl (not shown). As expected from the bowl-repressing function of lines, the overexpression of lines along the margin phenocopies the loss of bowl. Nevertheless, the overexpression of bowl in other eye disc regions is not sufficient to induce hh. This suggests that, in regions other than the margin, either the levels of lines are too high to be overcome by bowl or bowl requires other factors to induce hh, or both (Bras-Pereira, 2006).

drm and odd are expressed together along the posterior disc margin-Pe, and drm (at least) is required for Bowl stabilization in leg discs. Nevertheless, the removal of neither drm nor odd function alone results in retinal defects. odd and drm may act redundantly during leg segmentation and this may also be the case in the eye margin. To test this, clones were induced of DfdrmP2, a deficiency that deletes drm, sob and odd, plus other genes. When DfdrmP2 clones affect the margin, the adjacent retina fails to differentiate, suggesting that drm and odd (and perhaps sob, for which no single mutation is available) act redundantly to promote bowl activity at the margin (although the possibility that other genes uncovered by this deficiency also contribute to the phenotype cannot be excluded). To test the function of each of these genes, drm, odd and sob were expressed in cell clones elsewhere in the eye disc. Only the overexpression of drm or odd induced ectopic retinogenesis, and this was restricted to the region immediately anterior to the MF, which is already eye committed. Interestingly, bowl is also expressed in this region of L3 discs. The retina-inducing ability of drm requires bowl, because retinogenesis is no longer induced in drm-expressing clones that simultaneously lack bowl function. Therefore, it seems that in the eye, drm (and very likely also odd) also promotes bowl function (Bras-Pereira, 2006).

The expression of hh or activation of its pathway anterior to the furrow is sufficient to generate ectopic retinal differentiation. Since (1) bowl is required for hh expression at the margin, (2) this hh expression is largely coincident with that of odd and drm, and (3) drm (and possibly odd) functionally interacts with bowl, whether drm- and odd-expressing clones induced the expression of hh was examined. In both types of clones hh expression is turned on autonomously, as detected with hh-Z, which would thus be responsible for the ectopic retinogenesis observed. That the normal drm/odd/bowl-expressing margin does not differentiate as eye could be explained if margin cells lack certain eye primordium-specific factors (Bras-Pereira, 2006).

These results indicate that the expression of odd and drm defines during L2 the region of the bowl-expressing margin that is competent to induce retinogenesis. How is their expression controlled? wingless (wg) is expressed in the anterior margin, where it prevents the start of retinal differentiation. drm/odd are complementary to wg (monitored by wgZ) during early L3, when retinal differentiation is about to start, and also during later stages. In addition, when wg expression is reduced during larval life in wgCX3 mutants, drm transcription is extended all the way anteriorly. This extension precedes and prefigures the ectopic retinal differentiation that, in these mutants, occurs along the dorsal margin. Therefore, wg could repress anterior retinal differentiation by blocking the expression of odd genes in the anterior disc margin, in addition to its known role in repressing dpp expression and signaling (Bras-Pereira, 2006).

Interestingly, the onset of retinogenesis in L3 is delayed relative to the initiation of the expression of drm/odd and hh in L1-2. This delay can be explained in three, not mutually exclusive, ways. (1) The relevant margin factors (i.e. drm/odd, hh) might be in place early, but the eye primordium might become competent to respond to them later. In fact, wg expression domain has to retract anteriorly as the eye disc grows, under Notch signaling influence, to allow the expression of eye-competence factors. (2) Building up a concentration of margin factors sufficient to trigger retinogenesis might require some time. In fact, the activity of the Notch pathway along the prospective dorsoventral border is required to reinforce hh transcription at the firing point. (3) Other limiting factors might exist whose activity becomes available only during L3. Such a factor might be the EGF receptor pathway, which is involved in the triggering and reincarnation of the furrow along the margins during L3 (Bras-Pereira, 2006).

Organization of the peripheral fly eye: the roles of Snail family transcription factors in peripheral retinal apoptosis

The periphery of the fly eye contains a number of concentrically arranged cellular specializations that are induced by Wingless (Wg) signaling from the surrounding head capsule (HC). One of these is the pigment rim (PR), which is a thick layer of pigment cells that lies directly adjacent to the HC and completely circumscribes the rest of the retina. Many of the cells of the PR are derived from presumptive pigment cells that previously surrounded peripheral ommatidia that subsequently died. This study describes the Wg-elicited expression of Snail family transcription factors in the eye periphery that directs the ommatidial death and subsequent PR formation. These transcription factors are expressed only in a subset of the ommatidial cells not including the photoreceptors. Yet, the photoreceptors die and, thus, a non-autonomous death signal is released from the Snail-family-expressing cells that direct the death of the photoreceptors. In addition, Wg also elicits a similar peripheral expression of Notum, an enzyme that limits the extent of Wg signaling. Furthermore, a later requirement is described for Snail family proteins in the 2° and 3° pigment cells throughout the main body of the eye (Lim, 2006).

Wg signaling regulates the expression of Snail family genes, and a number of TCF-binding sites have been identified in the region of the three Snail genes, which is consistent with, but not proof of a direct regulation by the Wg transduction pathway. In mammalian systems, it had been shown that Snail transcription is elicited by the inhibition of glycogen synthase kinase-3 (GSK-3) which represses Snail expression by inhibiting the transcriptional activity of NFkappaB on the Snail promoter. In addition, GSK-3 can phosphorylate Snail at two consensus motifs, one for protein degradation (site I) and the other for subcellular localization (site II). Thus, in mammalian systems, Wnt signaling regulates Snail gene activity both at the level of the transcript and the protein (Lim, 2006).

The apoptotic removal of the most peripheral ring of developing ommatidia releases the surviving surrounding pigment cells to join and thicken the PR. Ectopic expression of Snail family proteins mimics the ommatidial death that is engendered by Wg expression, and loss of these proteins prevents the normal Wg-dependent removal of the peripheral ommatidia and consequently disrupts the PR. The Snail family transcription factors thus appear to direct the death of the peripheral ommatidia and development of the PR. However, within the peripheral ommatidia these proteins are expressed only in the cone cells -- they are absent from the photoreceptors (R cells) and the 1° pigment cells. They are also present in the pigment cells surrounding the ommatidia. This expression profile raises a number of points (Lim, 2006).

As the Snail family proteins are transcription factors, then the death signal is probably under their transcription control, but the molecular nature of the signal remains unknown (Lim, 2006).

As the R cells and 1° pigment cells are directed to apoptosis by the expression of Snail family proteins in other cells, then there is non-autonomous death induction. The non-autonomous initiation of death is envisaged in two possible forms. In the first model, the Snail-expressing cells sequester a survival factor that is thereby denied to other cells. Given that the cone cells express the Snail proteins but still die, this seems unlikely. The second model is that there is a factor released by Snail-expressing cells that directs the death of the ommatidial cells. The cells expressing the death factor may be the peripheral cone cells, the surrounding pigment cells or both. The second model is favored and the remainder of this discussion assumes this is correct with appropriate reservation (Lim, 2006).

The pigment cells surrounding the peripheral ommatidia are impervious to the death signal. One possibility is that the death signal is presented exclusively by the peripheral cone cells and only to the cells of the ommatidia (including themselves, and R cells and 1° cells) -- not to the surrounding pigment cells. The cone cells die before the R cells (the time of death of the 1° cells was not examined), and if the cone cells were the source of the death signal then they would probably receive the signal first. Alternatively, the pigment cells may release the death signal (secreted by themselves or the cone cells) but are programmed not to respond (Lim, 2006).

Only the cone cells of the peripheral ommatidia express Snail family proteins (and Wg and Notum) in response to Wg signaling from the HC -- the R cells and 1° cells do not. This probably represents a predisposition of the cone cells to respond to the Wg signal resulting from the selective expression of cone cell specific factors; Cut, for example, is a homeodomain transcription factor restricted to the cone cells at this stage (Lim, 2006).

The finding that Snail transcription factors promote death in Drosophila eye periphery is in contrast to their anti-apoptotic roles in other systems. For example in C. elegans, the Snail-like CES-1 (cell death specification) protein blocks death of the NSM sister cells during embryogenesis. In vertebrates, Slug (Snail2) is aberrantly upregulated by the E2F-HLF oncoprotein in some leukemias, leading to increased cell survival. Mammalian Snail has also been shown to confer resistance to cell death induced by the withdrawal of survival factors in cell cultures. However, in the fly eye a non-autonomous effect of Snail transcription family members in apoptosis is described, suggesting that a different molecular pathway is regulated from those of the autonomous examples above (Lim, 2006).

The death of the peripheral ommatidia appears to serve two functions - it removes these degenerate optical units and it supplies cells for the PR that optically insulate the entire eye. With regard to the PR, there are two sources of cells. First there is the thin layer of pigment cells that circumscribes the entire pupal eye and second there are the later cells, originally associated with the moribund ommatidia, that eventually incorporate into the existing PR to thicken it. Both aspects of PR formation appear to be under Wg signaling control. During the larval phase, the Hedgehog (Hh) morphogenetic wave sweeps the presumptive retina, triggering the ommatidial differentiation process. However, Wg is expressed in the flanking HC which inhibits the inductive mechanism. Thus, the larval retinal tissue directly adjacent to the HC does not undergo ommatidial differentiation. The 2° and 3° pigment cell fate appears to be the ground state of the retinal tissue, and thus the cells directly adjacent to the HC are destined to the pigment cell fate. Later in the pupa, Wg signaling triggers the death of the peripheral ommatidia and releases their pigment cells to join the PR and increase its thickness (Lim, 2006).

The expression of both Wg and Notum (its antagonist) by the cone cells of the peripheral ommatidia is interesting. It may suggest that high levels of Wg expression are required in the peripheral cone cells, but that the diffusion of this cone-cell derived Wg needs to be tightly contained. For example, in the model above where the death signal is provided by the peripheral cone cells, high levels of Wg may be needed to trigger sufficient levels of the apoptotic signal but any diffusion of the high levels of Wg would disturb other aspects of the peripheral patterning (Lim, 2006).

In the absence of Notum, the effects of Wg signaling spread approximately one more ommatidial row into the eye periphery. This relatively mild phenotype suggests that there could be redundant mechanisms restricting the movement of Wg gradient at the eye margin. In Drosophila wing disc, the Wg receptor Drosophila Frizzled2 (Fz2) stabilizes Wg and allows it to reach cells far from its site of synthesis. Wg signaling represses Fz2 expression, creating a gradient of decreasing Wg stability towards the D/V boundary. This might also be the case in the eye periphery, where Wg signaling, in addition to activating Notum, might also represses Fz2 to limit the extent of Wg diffusion (Lim, 2006).

Snail family gene expression in the 2° and 3° pigment cells appears to be under two different control mechanisms; in the peripheral regions it is activated by Wg signaling, but in the main body of the eye it is not. Furthermore, the genes of the Snail complex appear functionally redundant in the periphery but not in the main body of the eye. Here, the phenotypes of esg clones are as strong as those of the mutations in all three genes. This may be explained by differential regulation of the gene promoters in the two positions. For example, in the main body of the eye, Esg expression in the 2° and 3° pigment cells may activate expression of the two other genes, but in the periphery, Wg signaling directly activates each of the genes, with no cross-regulation between them. The majority of studies on the specification of the main body 2° and 3° pigment cells have focused on the mechanism of weeding out the surplus interommatidial cells which occurs between 18 hours and 36 hours APF, but little is known about their subsequent maturation. The data showed that Esg is expressed in the interommatidial pigment cells after the cell pruning mechanism, but before any sign of morphological differentiation. In the esg mutants, the 2° and 3° pigment cells do not undergo correct apical constriction, indicating that these cells are either developmentally delayed compared with their wild-type counterparts or are blocked in their maturation. If the cells are simply developmentally delayed, they should mature over time, but esg mutant clones in the adult eye show degenerate or lost 2° and 3° pigment cells. Thus, Esg appears required for the appropriate maturation/survival of the 2° and 3° pigment cells. What happens to the esg mutant pigment cells after the point when they fail to undergo apical restriction (whether they delaminate or die/degenerate in place) remains to be investigated (Lim, 2006).

Wingless targets in the brain

The morphogenesis of specialized structures within the CNS relies on the nonautonomous activity of cell populations that play the role of organizers. In the Drosophila visual system, cells on the dorsal and ventral margins of the developing visual cortex express the Wnt family member Wingless (Wg) and the TGF-beta Decapentaplegic (Dpp). The activity of these morphogens in establishing cortical cell fates sets the stage for the guidance of photoreceptor axons to their retinotopic destinations in the Drosophila brain. One role for Wg in cortical development is to induce and maintain the expression of Dpp, a key step in the assignment of dorsoventral cell identities. Dpp is induced early in cortical development, shortly after the onset of Wg expression in a few dorsal and ventral margin cells, and is maintained by Wg activity until at least the time of retinal axon pathfinding. Wg is a developmental signal in many different tissues, and acts by regulating different target gene sets to elicit a constellation of different cell fates. Wingless-controlled targets include distal-less and vestigial in the wing, engrailed in the embryonic ectoderm, labial in the gut, and sloppy-paired in the embryonic CNS. Conversely, Dpp belongs to a Hedgehog-controlled circuit in the wing (Song, 2000 and references therein).

A regulatory mechanism is described that relays Wg signal reception to the tissue-specific expression of target genes in the visual cortex. In a screen for mutants in which photoreceptor axons project aberrantly to their destinations in the brain, a mutation in combgap was discovered. Retinal axon navigation defects in combgap animals are due to the role of cg in the establishment of cortical cell identity. cg represses the expression of Wg target genes in a positionally restricted manner in the visual cortex. wg+ induction of its cortical cell targets occurs via the downregulation of cg. Combgap is thus a tissue-specific relay between Wingless and its target genes for the determination of cell fate in the visual cortex (Song, 2000).

A combgap mutation was recovered in a screen for mutants with aberrations in retinal axon projections. On the basis of its effects on target region gene expression and the outcome of mosaic analysis, it is evident that a role for combgap in the specification of cortical cell identity underlies its requirement for the establishment of retinotopic connectivity in the visual system. In cg loss of function animals, three markers under wg+ control are expressed in expanded dorsal and ventral portions of the retinal axon target field. The requirement for cg to repress the markers within these domains is autonomous. The lamina midline region, however, appears phenotypically normal in homozygous or mosaic cg animals. This positionally restricted requirement for cg+ activity is correlated with the pattern of cg expression, since cg is not expressed in the midline region where it is not required. Since wg+ misexpression is sufficient to induce wg+-dependent markers in the midline region, another regulatory system must control these markers there. Hence, the consequences of wg signal reception at different dorsoventral positions within the cortical precursor field would appear to involve a set of regulatory molecules that divide the cortex into specific domains for pattern formation (Song, 2000).

The constellation of genes under Wingless control displays considerable tissue specificity. Wingless-controlled targets include Distal-less and vestigial in the wing, engrailed in the embryonic ectoderm, and sloppy-paired in the embryonic CNS. Though Dpp and Omb belong to a Hedgehog-controlled circuit in the wing, they are under Wg control in the visual cortices of the brain. With respect to the control of cell fate, Wg signal transduction apparently follows a canonical pathway from a pair of redundant receptors at the cell surface to the cytoplasmic control of Armadillo stability and nuclear translocation. This raises the question of how the tissue specificity of wg target gene expression is achieved (Song, 2000).

The observations that cg regulates dpp, optimotor blind and aristaless in the visual cortex place cg in a second tier of regulation, as a component of a tissue-specific relay mechanism between the Wg signal transduction pathway and the target genes that are wg dependent in visual system cortical cells. The evidence in support of this hypothesis is as follows: (1) epistasis analysis with the wg pathway negative regulator Axn places the requirement for cg downstream of the cytoplasmic complex that includes APC, GSK-beta, and Armadillo; (2) the induction of at least three downstream effectors of wg+ activity is mediated by negative regulation of cg expression -- cg expression is reduced in the dorsal and ventral domains of the cortical lamina where these wg target genes are expressed and ectopic cg expression blocks wg target gene expression within these domains; (3) ectopic wg+ clones repress cg expression, yielding Cg-negative domains in which wg target genes are ectopically expressed. The presence of consensus Pangolin binding sites in the first intron of cg suggests cg may be a direct target of Wg signal transduction. How the Armadillo/Pangolin complex might participate in the negative regulation of cg is unclear. Cg might act by binding directly to wg target gene regulatory elements as a transcriptional repressor (Song, 2000).

The pattern of Combgap expression in the developing visual ganglia was examined by in situ hybridization and by staining with an anti-Cg antiserum. Both analyses gave similar results. Cg is expressed most strongly in dorsal and ventral regions of the optic ganglia, and weakly or not at all in the midline region. cg transcript and protein are reduced in the vicinity of the wg+ domains. The absence of Cg in the midline region is consistent with the lack of phenotypic effects of cg mutations in this region. Since ectopic wg+ activity is sufficient to induce the ectopic expression of wg target genes in the midline region, it is supposed that other factors are responsible for wg target gene regulation there. The reduction of cg expression found in the vicinity of wg+ target gene expression is consistent with the notion that wg+ induction of its optic lobe targets occurs via the downregulation of Cg expression (Song, 2000).

These observations along with the role of Cg as a negative regulator of wg target genes suggest that Cg expression might be regulated by wg+. Consistent with this notion, three consensus dTCF binding sites were identified within the first intron of the cg locus. To determine whether Cg expression is indeed under wg+ control, animals were generated carrying ectopic wg+ clones. The wg+ clones resulting from recombination between the repeated FRT sites were visualized by their failure to express the CD2 marker. The presence of ectopic wg+-expressing cells could also be inferred by the local nonautonomous induction of the target gene omb. The induction of omb by ectopic wg+ expression coincides with a reduction in Cg expression. This effect of wg+ is nonautonomous, as both the induction of Omb and the reduction of Cg expression have been found to extend beyond the boundary of marked wg+ clones. Cg expression thus appears to be under the nonautonomous control of wg+ activity (Song, 2000).

Table of contents

wingless continued: Biological Overview | Evolutionary Homologs | Transcriptional regulation | Protein Interactions | mRNA Transport | Developmental Biology | Effects of Mutation | References

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