Interactive Fly, Drosophila

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).

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, 1999 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, 1999 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, 1999).

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, 1999 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, 1999).

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, 1999).

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, 1999).

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, 1999).

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).

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).

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