The role of Wingless signaling in establishing the anteroposterior and dorsoventral axes of the eye disc

The posteriorly expressed signaling molecules Hedgehog and Decapentaplegic drive photoreceptor differentiation in the Drosophila eye disc, while at the anterior lateral margins Wingless expression blocks ectopic differentiation. We show here that mutations in axin prevent photoreceptor differentiation and lead to tissue overgrowth and that both these effects are due to ectopic activation of the Wingless pathway. In addition, ectopic Wingless signaling causes posterior cells to take on an anterior identity, reorienting the direction of morphogenetic furrow progression in neighboring wild-type cells. We also show that signaling by Decapentaplegic and Hedgehog normally blocks the posterior expression of anterior markers such as Eyeless. Wingless signaling is not required to maintain anterior Eyeless expression and in combination with Decapentaplegic signaling can promote its downregulation, suggesting that additional molecules contribute to anterior identity. Along the dorsoventral axis of the eye disc, Wingless signaling is sufficient to promote dorsal expression of the Iroquois gene mirror, even in the absence of the upstream factor pannier. However, Wingless signaling does not lead to ventral mirror expression, implying the existence of ventral repressors.     

The canonical Wg and JNK signaling cascades collaborate to promote both dorsal closure and ventral patterning

Elaboration of the Drosophila body plan depends on a series of cell-identity decisions and morphogenetic movements regulated by intercellular signals. For example, Jun N-terminal kinase signaling regulates cell fate decisions and morphogenesis during dorsal closure, while Wingless signaling regulates segmental patterning of the larval cuticle via Armadillo. wingless or armadillo mutant embryos secrete a lawn of ventral denticles; armadillo mutants also exhibit dorsal closure defects. We found that mutations in puckered, a phosphatase that antagonizes Jun N-terminal kinase, suppress in a dose-sensitive manner both the dorsal and ventral armadillo cuticle defects. Furthermore, we found that activation of the Jun N-terminal kinase signaling pathway suppresses armadillo-associated defects. Jun N-terminal kinase signaling promotes dorsal closure, in part, by regulating decapentaplegic expression in the dorsal epidermis. We demonstrate that Wingless signaling is also required to activate decapentaplegic expression and to coordinate cell shape changes during dorsal closure. Together, these results demonstrate that MAP-Kinase and Wingless signaling cooperate in both the dorsal and ventral epidermis, and suggest that Wingless may activate both the Wingless and the Jun N-terminal kinase signaling cascades.     

Segment boundary formation in Drosophila embryos

In Drosophila embryos, segment boundaries form at the posterior edge of each stripe of engrailed expression. We have used an HRP-CD2 transgene to follow by transmission electron microscopy the cell shape changes that accompany boundary formation. The first change is a loosening of cell contact at the apical side of cells on either side of the incipient boundary. Then, the engrailed-expressing cells flanking the boundary undergo apical constriction, move inwards and adopt a bottle morphology. Eventually, grooves regress, first on the ventral side, then laterally. We noted that groove formation and regression are contemporaneous with germ band retraction and shortening, respectively, suggesting that these rearrangements could also contribute to groove morphology. The cellular changes accompanying groove formation require that Hedgehog signalling be activated, and, as a result, a target of Ci expressed, at the posterior of each boundary (obvious targets like stripe and rhomboid appear not to be involved). In addition, Engrailed must be expressed at the anterior side of each boundary, even if Hedgehog signalling is artificially maintained. Thus, there are distinct genetic requirements on either side of the boundary. In addition, Wingless signalling at the anterior of the domains of engrailed (and hedgehog) expression represses groove formation and thus ensures that segment boundaries form only at the posterior.     

Fate map of the distal portion of Drosophila proboscis as inferred from the expression and mutations of basic patterning genes

The late-third-instar labial disc is comprised of two disc-proper cell layers, one representing mainly the ventral half of the anterior compartment (L-layer) and the other, the dorsal half of the anterior compartment and most, if not all, of the posterior compartment (M-layer). In the L-layer, Distal-less represses homothorax whereas no Distal-less-dependent homothorax repression occurs in the M-layer where Distal-less is coexpressed with homothorax. In wild-type labial discs, clawless, one of the two homeobox genes expressed in distal cells receiving maximum (Decapentaplegic+Wingless) signaling activity in leg and antennal discs, is specifically repressed by proboscipedia. A fate map, inferred from data on basic patterning gene expression in larval and pupal stages and mutant phenotypes, indicates the inner surface of the labial palpus, which includes the pseudotracheal region, to be a derivative of the distal portion of the M-layer expressing wingless, patched, Distal-less and homothorax. The outer surface of the labial palpus with more than 30 taste bristles derives from an L-layer area consisting of dorsal portions of the anterior and posterior compartments, each expressing Distal-less. Our analysis also indicates that, in adults and pupae, the anterior-posterior boundary, dividing roughly equally the outer surface of the distiproboscis, runs along the outer circumference of the inner surface of distiproboscis.     

The glypican Dally-like is required for Hedgehog signalling in the embryonic epidermis of Drosophila

The Drosophila genes dally and dally-like encode glypicans, which are heparan sulphate proteoglycans anchored to the cell membrane by a glycosylphosphatidylinositol link. Genetic studies have implicated Dally and Dally-like in Wingless signalling in embryos and imaginal discs. Here, we test the signalling properties of these molecules in the embryonic epidermis. We demonstrate that RNA interference silencing of dally-like, but not dally, gives a segment polarity phenotype identical to that of null mutations in wingless or hedgehog. Using heterologous expression in embryos, we uncoupled the Hedgehog and Wingless signalling pathways and found that Dally-like and Dally, separately or together, are not necessary for Wingless signalling. Dally-like, however, is strictly necessary for Hedgehog signal transduction. Epistatic experiments show that Dally-like is required for the reception of the Hedgehog signal, upstream or at the level of the Patched receptor.     

Notch and Wingless Regulate Expression of Cuticle Patterning Genes

The cell surface receptor Notch is required during Drosophila embryogenesis for production of epidermal precursor cells. The secreted factor Wingless is required for specifying different types of cells during differentiation of tissues from these epidermal precursor cells. The results reported here show that the full-length Notch and a form of Notch truncated in the amino terminus associate with Wingless in S2 cells and in embryos. In S2 cells, Wingless and the two different forms of Notch regulate expression of Dfrizzled 2, a receptor of Wg; hairy, a negative regulator of achaete expression; shaggy, a negative regulator of engrailed expression; and patched, a negative regulator of wingless expression. Analyses of expression of the same genes in mutant N embryos indicate that the pattern of gene regulations observed in vitro reflects regulations in vivo. These results suggest that the strong genetic interactions observed between Notch and wingless genes during development of Drosophila is at least partly due to regulation of expression of cuticle patterning genes by Wingless and the two forms of Notch.     

Hedgehog signalling is required for correct anteroposterior patterning of the zebrafish otic vesicle

Currently, few factors have been identified that provide the inductive signals necessary to transform the simple otic placode into the complex asymmetric structure of the adult vertebrate inner ear. We provide evidence that Hedgehog signalling from ventral midline structures acts directly on the zebrafish otic vesicle to induce posterior otic identity. We demonstrate that two strong Hedgehog pathway mutants, chameleon (con(tf18b)) and slow muscle omitted (smu(b641)) exhibit a striking partial mirror image duplication of anterior otic structures, concomitant with a loss of posterior otic domains. These effects can be phenocopied by overexpression of patched1 mRNA to reduce Hedgehog signalling. Ectopic activation of the Hedgehog pathway, by injection of sonic hedgehog or dominant-negative protein kinase A RNA, has the reverse effect: ears lose anterior otic structures and show a mirror image duplication of posterior regions. By using double mutants and antisense morpholino analysis, we also show that both Sonic hedgehog and Tiggy-winkle hedgehog are involved in anteroposterior patterning of the zebrafish otic vesicle.     

Determination of cell fate along the anteroposterior axis of the Drosophila ventral midline

The Drosophila ventral midline has proven to be a useful model for understanding the function of central organizers during neurogenesis. The midline is similar to the vertebrate floor plate, in that it plays an essential role in cell fate determination in the lateral CNS and also, later, in axon pathfinding. Despite the importance of the midline, the specification of midline cell fates is still not well understood. Here, we show that most midline cells are determined not at the precursor cell stage, but as daughter cells. After the precursors divide, a combination of repression by Wingless and activation by Hedgehog induces expression of the proneural gene lethal of scute in the most anterior midline daughter cells of the neighbouring posterior segment. Hedgehog and Lethal of scute activate Engrailed in these anterior cells. Engrailed-positive midline cells develop into ventral unpaired median (VUM) neurons and the median neuroblast (MNB). Engrailed-negative midline cells develop into unpaired median interneurons (UMI), MP1 interneurons and midline glia.     

Segmenting the fly embryo: logical analysis of the role of the segment polarity cross-regulatory module

Initially activated by the pair-rule genes, the expression patterns of the segment polarity genes engrailed and wingless become consolidated through inter-cellular interactions between juxtaposed cells. We delineate a logical model focusing on a dozen molecular components at the core of the regulatory network controlling this process. Our model leads to the following conclusions: (1) the pair-rule signals, which activate engrailed and wingless genes independently of each other, need to be operative until the inter-cellular circuit involving these two genes is functional. This implies that the pair-rule pattern is instrumental both in determining the activation of the genes engrailed and wingless in rows of adjacent cells, and in consolidating these expression patterns; (2) the consolidation of engrailed and wingless expression patterns requires the simultaneous activation of both autocrine and paracrine Wingless-pathways, and the Hedgehog pathway; (3) protein kinase A plays at least two roles through the phosphorylation of Cubitus interruptus, the effector molecule of the Hedgehog signalling pathway and (4) the roles of Sloppy-paired and Naked in the delineation of the engrailed and wingless expression domains are emphasized as being important for segmental boundary formation. Moreover, the application of an original computational method leads to the delineation of a subset of crucial regulatory circuits enabling the coexistence of specific expression states at the cellular level, as well as specific combination of cellular states inter-connected through Wingless and Hedgehog signalling. Finally, the simulation of altered expressions of segment polarity genes leads to results consistent with the published data.     

hedgehog and engrailed: pattern formation and polarity in the Drosophila abdomen

Like the Drosophila embryo, the abdomen of the adult consists of alternating anterior (A) and posterior (P) compartments. However the wing is made by only part of one A and part of one P compartment. The abdomen therefore offers an opportunity to compare two compartment borders (A/P is within the segment and P/A intervenes between two segments), and ask if they act differently in pattern formation. In the embryo, abdomen and wing P compartment cells express the selector gene engrailed and secrete Hedgehog protein whilst A compartment cells need the patched and smoothened genes in order to respond to Hedgehog. We made clones of cells with altered activities of the engrailed, patched and smoothened genes. Our results confirm (1) that the state of engrailed, whether 'off' or 'on', determines whether a cell is of A or P type and (2) that Hedgehog signalling, coming from the adjacent P compartments across both A/P and P/A boundaries, organises the pattern of all the A cells. We have uncovered four new aspects of compartments and engrailed in the abdomen. First, we show that engrailed acts in the A compartment: Hedgehog leaves the P cells and crosses the A/P boundary where it induces engrailed in a narrow band of A cells. engrailed causes these cells to form a special type of cuticle. No similar effect occurs when Hedgehog crosses the P/A border. Second, we look at the polarity changes induced by the clones, and build a working hypothesis that polarity is organised, in both compartments, by molecule(s) emanating from the A/P but not the P/A boundaries. Third, we show that both the A and P compartments are each divided into anterior and posterior subdomains. This additional stratification makes the A/P and the P/A boundaries fundamentally distinct from each other. Finally, we find that when engrailed is removed from P cells (of, say, segment A5) they transform not into A cells of the same segment, but into A cells of the same parasegment (segment A6).     

Roles of wingless in patterning the larval epidermis of Drosophila.

The larval epidermis of Drosophila shows a stereotyped segmentally repeating pattern of cuticular structures. Mutants deficient for the wingless gene product show highly disrupted patterning of the larval cuticle. We have manipulated expression of the wg gene product to assess its role in this patterning process. We present evidence for four distinct phases of wg function in epidermal cells: (1) an early requirement in engrailed-expressing cells to establish and maintain stable expression of en, (2) a discrete period when wg and en gene products act in concert to generate positional values in the anterior portion of the ventral segment and all values of the dorsal and lateral epidermis, (3) a progressive function (dependent on prior interaction with the en-expressing cells) in conferring positional values to cells within the posterior portion of the segment, and (4) a late continuous requirement for maintaining some ventral positional values.     

Evidence for Wg-independent tergite boundary formation in the millipede Glomeris marginata

The correlation between dorsal and ventral segmental units in diplopod myriapods is complex and disputed. Recent results with engrailed (en), hedgehog (hh), wingless (wg), and cubitus-interruptus (ci) have shown that the dorsal segments are patterned differently from the ventral segments. Ventrally, gene expression is compatible with the classical autoregulatory loop known from Drosophila to specify the parasegment boundary. In the dorsal segments, however, this Wg/Hh autoregulatory loop cannot be present because the observed gene expression patterns argue against the involvement of Wg signalling. In this paper, we present further evidence against an involvement of Wg signalling in dorsal segmentation and propose a hypothesis about how dorsal segmental boundaries may be controlled in a wg-independent way. We find that (1) the Notum gene, a modulator of the Wg gradient in Drosophila, is not expressed in the dorsal segments. (2) The H15/midline gene, a repressor of Wg action in Drosophila, is not expressed in the dorsal segments, except for future heart tissue. (3) The patched (ptc) gene, which encodes a Hh receptor, is strongly expressed in the dorsal segments, which is incompatible with Wg-Hh autoregulation. The available data suggest that anterior-posterior (AP) boundary formation in dorsal segments could instead rely on Dpp signalling rather than Wg signalling. We present a hypothesis that relies on Hh-mediated activation of Dpp signalling and optomotor-blind (omb) expression to establish the dorsal AP boundary (the future tergite boundary). The proposed mechanism is similar to the mechanism used to establish the AP boundary in Drosophila wings and ventral pleura.     

Control of Drosophila eye specification by Wingless signalling

Organ formation requires early specification of the groups of cells that will give rise to specific structures. The Wingless protein plays an important part in this regional specification of imaginal structures in Drosophila, including defining the region of the eye-antennal disc that will become retina. We show that Wingless signalling 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 signalling leads to the repression of these genes and the loss of eyes, whereas loss of Wingless signalling 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, we find that under conditions of excess Wingless signalling, eye tissue is transformed not only into head cuticle but also into a variety of inappropriate structures.