Wingless modulates the effects of dominant negative notch molecules in the developing wing of Drosophila

The development and patterning of the wing in Drosophila relies on a sequence of cell interactions molecularly driven by a number of ligands and receptors. Genetic analysis indicates that a receptor encoded by the Notch gene and a signal encoded by the wingless gene play a number of interdependent roles in this process and display very strong functional interactions. At certain times and places, during wing development, the expression of wingless requires Notch activity and that of its ligands Delta and Serrate. This has led to the proposal that all the interactions between Notch and wingless can be understood in terms of this regulatory relationship. Here we have tested this proposal by analysing interactions between Delta- and Serrate-activated Notch signalling and Wingless signalling during wing development and patterning. We find that the cell death caused by expressing dominant negative Notch molecules during wing development cannot be rescued by coexpressing Nintra. This suggests that the dominant negative Notch molecules cannot only disrupt Delta and Serrate signalling but can also disrupt signalling through another pathway. One possibility is the Wingless signalling pathway as the cell death caused by expressing dominant negative Notch molecules can be rescued by activating Wingless signalling. Furthermore, we observe that the outcome of the interactions between Notch and Wingless signalling differs when we activate Wingless signalling by expressing either Wingless itself or an activated form of the Armadillo. For example, the effect of expressing the activated form of Armadillo with a dominant negative Notch on the patterning of sense organ precursors in the wing resembles the effects of expressing Wingless alone. This result suggests that signalling activated by Wingless leads to two effects, a reduction of Notch signalling and an activation of Armadillo.     

Notch responds differently to Delta and Wingless in cultured Drosophila cells

Notch, a cell surface receptor, is required for producing different types of cells during development of Drosophila melanogaster. Notch activates expression of one set of genes in response to ligand Delta and another set of genes in response to ligand Wingless. The means by which Notch initiates these different intracellular activities was examined in this study. Cultured cells expressing Notch were treated with Delta or Wingless, and the effect on Notch was examined by Western blotting. Treatment of cells with Delta resulted in accumulation of approximately 120-kDa Notch intracellular domain molecules in the cytoplasmic fraction. This form of Notch did not accumulate in cells treated with Wingless, but the approximately 350-kDa full-length Notch molecules accumulated. These results indicate that N responds differently to binding by Delta and Wingless, and suggest that although the Delta signal is transduced by the Notch intracellular domain released from the plasma membrane, the Wingless signal is transduced by the Notch intracellular domain associated with the plasma membrane.     

Multiple signalling pathways establish cell fate and cell number in Drosophila malpighian tubules

A unique cell, the tip mother cell, arises in the primordium of each Drosophila Malpighian tubule by lateral inhibition within a cluster of achaete-expressing cells. This cell maintains achaete expression and divides to produce daughters of equivalent potential, of which only one, the tip cell, adopts the primary fate and continues to express achaete, while in the other, the sibling cell, achaete expression is lost (M. Hoch et al., 1994, Development 120, 3439-3450). In this paper we chart the mechanisms by which achaete expression is differentially maintained in the tip cell lineage to stabilise cell fate. First, wingless is required to maintain the expression of achaete in the tubule primordium so that wingless mutants lack tip cells. Conversely, increasing wingless expression results in the persistence of achaete expression in the cell cluster. Second, Notch signalling is restricted by the asymmetric segregation of Numb, as the tip mother cell divides, so that achaete expression is maintained only in the tip cell. In embryos mutant for Notch tip cells segregate at the expense of sibling cells, whereas in numb neither daughter cell adopts the tip cell fate resulting in tubules with two sibling cells. Conversely, when numb is overexpressed two tip cells segregate and tubules have no sibling cells. Analysis of cell proliferation in the developing tubules of embryos lacking Wingless after the critical period for tip cell allocation reveals an additional requirement for wingless for the promotion of cell division. In contrast, alteration in the expression of numb has no effect on the final tubule cell number.     

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.     

Armadillo/beta-catenin-dependent Wnt signalling is required for the polarisation of epidermal cells during dorsal closure in Drosophila

At the end of germband retraction, the dorsal epidermis of the Drosophila embryo exhibits a discontinuity that is covered by the amnioserosa. The process of dorsal closure (DC) involves a coordinated set of cell-shape changes within the epidermis and the amnioserosa that result in epidermal continuity. Polarisation of the dorsal-most epidermal (DME) cells in the plane of the epithelium is an important aspect of DC. The DME cells of embryos mutant for wingless or dishevelled exhibit polarisation defects and fail to close properly. We have investigated the role of the Wingless signalling pathway in the polarisation of the DME cells and DC. We find that the beta-catenin-dependent Wingless signalling pathway is required for polarisation of the DME cells. We further show that although the DME cells are polarised in the plane of the epithelium and present polarised localisation of proteins associated with the process of planar cell polarity (PCP) in the wing, e.g. Flamingo, PCP Wingless signalling is not involved in DC.     

Novel Notch alleles reveal a Deltex-dependent pathway repressing neural fate.

BACKGROUND: The Notch receptor triggers a wide range of cell fate choices in higher organisms. In Drosophila, segregation of neural from epidermal lineages results from competition among equivalent cells. These cells express achaete/scute genes, which confer neural potential. During lateral inhibition, a single neural precursor is selected, and neighboring cells are forced to adopt an epidermal fate. Lateral inhibition relies on proteolytic cleavage of Notch induced by the ligand Delta and translocation of the Notch intracellular domain (NICD) to the nuclei of inhibited cells. The activated NICD, interacting with Suppressor of Hairless [Su(H)], stimulates genes of the E(spl) complex, which in turn repress the proneural genes achaete/scute. RESULTS: Here, we describe new alleles of Notch that specifically display loss of microchaetae sensory precursors. This phenotype arises from a repression of neural fate, by a Notch signaling distinct from that involved in lateral inhibition. We show that the loss of sensory organs associated with this phenotype results from a constitutive activation of a Deltex-dependent Notch-signaling event. These novel Notch alleles encode truncated receptors lacking the carboxy terminus of the NICD, which is the binding site for the repressor Dishevelled (Dsh). Dsh is known to be involved in crosstalk between Wingless and Notch pathways. CONCLUSIONS: Our results reveal an antineural activity of Notch distinct from lateral inhibition mediated by Su(H). This activity, mediated by Deltex (Dx), represses neural fate and is antagonized by elements of the Wingless (Wg)-signaling cascade to allow alternative cell fate choices.     

Son of Notch, a winged-helix gene involved in boundary formation in the Drosophila wing

A P element enhancer trap screen was conducted to identify genes involved in dorsal-ventral boundary formation in Drosophila. The son of Notch (son) gene was identified by the son(2205) enhancer trap insertion, which is a partial loss-of-function mutation. Based on son(2205) mutant phenotypes and genetic interactions with Notch and wingless mutations, we conclude that son participates in wing development, and functions in the Notch signaling pathway at the dorsal-ventral boundary in the wing. Notch signaling pathway components activate son enhancer trap expression in wing cells. son enhancer trap expression is regulated positively by wingless, and negatively by cut in boundary cells. Ectopic Son protein induces wingless and cut expression in wing discs. We hypothesize that there is positive feedback regulation of son by wingless, and negative regulation by cut at the dorsal-ventral boundary during wing development.     

Prepattern genes and signaling molecules regulate stripe expression to specify Drosophila flight muscle attachment sites

In Drosophila, muscles attach to epidermal tendon cells specified by the gene stripe (sr). Flight muscle attachment sites are prefigured on the wing imaginal disc by sr expression in discrete domains. We describe the mechanisms underlying the specification of these domains of sr expression. We show that the concerted activities of the wingless (wg), decapentaplegic (dpp) and Notch (N) signaling pathways, and the prepattern genes pannier (pnr) and u-shaped (ush) establish domains of sr expression. N is required for initiation of sr expression. pnr is a positive regulator of sr, and is inhibited by ush in this function. The Wg signal differentially influences the formation of different sr domains. These results identify the multiple regulatory elements involved in the positioning of Drosophila flight muscle attachment sites.     

Distinct functions of the Drosophila genes Serrate and Delta revealed by ectopic expression during wing development

 The Drosophila gene Serrate encodes a transmembrane protein with 14 epidermal growth factor-(EGF)-like repeats in its extracellular portion. It has been suggested to act as a signal in the developing wing from the dorsal side to induce the organising centre at the dorsal/ventral compartment boundary, which is required for growth and patterning of the wing. Ectopic expression of Serrate during wing development induces ectopic outgrowth of ventral wing tissue and the formation of an additional wing margin. Here we present data to suggest that both events are mediated by genes that are required for normal wing development, including Notch as receptor. In order for Serrate to elicit these responses the concomitant expression of wingless seems to be required. The lack of wings in flies devoid of Serrate function can be partially restored by Gal4-mediated expression of Serrate, whilst expression of wingless is not sufficient. Ectopic expression of Delta, which encodes a structurally very similar transmembrane protein with EGF-like repeats, provokes wing outgrowth and induction of a new margin under all conditions tested here, both on the dorsal and ventral side. Our data further suggest that Serrate can act as an activating ligand for the Notch receptor only under certain circumstances; it inhibits Notch function under other conditions. Received: 26 april 1996 / Accepted: 24 May 1996     

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.     

Differential requirements of the fused kinase for hedgehog signalling in the Drosophila embryo.

The two signalling proteins, Wingless and Hedgehog, play fundamental roles in patterning cells within each metamere of the Drosophila embryo. Within the ventral ectoderm, Hedgehog signals both to the anterior and posterior directions: anterior flanking cells express the wingless and patched Hedgehog target genes whereas posterior flanking cells express only patched. Furthermore, Hedgehog acts as a morphogen to pattern the dorsal cuticle, on the posterior side of cells where it is produced. Thus responsive embryonic cells appear to react according to their position relative to the Hedgehog source. The molecular basis of these differences is still largely unknown. In this paper we show that one component of the Hedgehog pathway, the Fused kinase accumulates preferentially in cells that could respond to Hedgehog but that Fused concentration is not a limiting step in the Hedgehog signalling. We present direct evidence that Fused is required autonomously in anterior cells neighbouring Hedgehog in order to maintain patched and wingless expression while Wingless is in turn maintaining engrailed and hedgehog expression. By expressing different components of the Hedgehog pathway only in anterior, wingless-expressing cells we could show that the Hedgehog signalling components Smoothened and Cubitus interruptus are required in cells posterior to Hedgehog domain to maintain patched expression whereas Fused is not necessary in these cells. This result suggests that Hedgehog responsive ventral cells in embryos can be divided into two distinct types depending on their requirement for Fused activity. In addition, we show that the morphogen Hedgehog can pattern the dorsal cuticle independently of Fused. In order to account for these differences in Fused requirements, we propose the existence of position-specific modulators of the Hedgehog response.     

Frizzled and Dfrizzled-2 function as redundant receptors for Wingless during Drosophila embryonic development.

In cell culture assays, Frizzled and Dfrizzled2, two members of the Frizzled family of integral membrane proteins, are able to bind Wingless and transduce the Wingless signal. To address the role of these proteins in the intact organism and to explore the question of specificity of ligand-receptor interactions in vivo, we have conducted a genetic analysis of frizzled and Dfrizzled2 in the embryo. These experiments utilize a small gamma-ray-induced deficiency that uncovers Dfrizzled2. Mutants lacking maternal frizzled and zygotic frizzled and Dfrizzled2 exhibit defects in the embryonic epidermis, CNS, heart and midgut that are indistinguishable from those observed in wingless mutants. Epidermal patterning defects in the frizzled, Dfrizzled2 double-mutant embryos can be rescued by ectopic expression of either gene. In frizzled, Dfrizzled2 mutant embryos, ectopic production of Wingless does not detectably alter the epidermal patterning defect, but ectopic production of an activated form of Armadillo produces a naked cuticle phenotype indistinguishable from that produced by ectopic production of activated Armadillo in wild-type embryos. These experiments indicate that frizzled and Dfrizzled2 function downstream of wingless and upstream of armadillo, consistent with their proposed roles as Wingless receptors. The lack of an effect on epidermal patterning of ectopic Wingless in a frizzled, Dfrizzled2 double mutant argues against the existence of additional Wingless receptors in the embryo or a model in which Frizzled and Dfrizzled2 act simply to present the ligand to its bona fide receptor. These data lead to the conclusion that Frizzled and Dfrizzled2 function as redundant Wingless receptors in multiple embryonic tissues and that this role is accurately reflected in tissue culture experiments. The redundancy of Frizzled and Dfrizzled2 explains why Wingless receptors were not identified in earlier genetic screens for mutants defective in embryonic patterning.     

Temporally dynamic response to Wingless directs the sequential elaboration of the proximodistal axis of the Drosophila wing

The Drosophila wing imaginal disc gives rise to three main regions along the proximodistal axis of the dorsal mesothoracic segment: the notum, proximal wing, and wing blade. Development of the wing blade requires the Notch and wingless signalling pathways to activate vestigial at the dorsoventral boundary. However, in the proximal wing, Wingless activates a different subset of genes, e.g., homothorax. This raises the question of how the downstream response to Wingless signalling differentiates between proximal and distal fate specification. Here, we show that a temporally dynamic response to Wingless signalling sequentially elaborates the proximodistal axis. In the second instar, Wingless activates genes involved in proximal wing development; later in the third instar, Wingless acts to direct the differentiation of the distal wing blade. The expression of a novel marker for proximal wing fate, zfh-2, is initially activated by Wingless throughout the "wing primordium," but later is repressed by the activity of Vestigial and Nubbin, which together define a more distal domain. Thus, activation of a distal developmental program is antagonistic to previously established proximal fate. In addition, Wingless is required early to establish proximal fate, but later when Wingless activates distal differentiation, development of proximal fate becomes independent of Wingless signalling. Since P-element insertions in the zfh-2 gene result in a revertable proximal wing deletion phenotype, it appears that zfh-2 activity is required for correct proximal wing development. Our data are consistent with a model in which Wingless first establishes a proximal appendage fate over notum, then the downstream response changes to direct the differentiation of a more distal fate over proximal. Thus, the proximodistal domains are patterned in sequence and show a distal dominance.     

Border of Notch activity establishes a boundary between the two dorsal appendage tube cell types

Boundaries establish and maintain separate populations of cells critical for organ formation. We show that Notch signaling establishes the boundary between two types of post-mitotic epithelial cells, the Rhomboid- and the Broad-positive cells. These cells will undergo morphogenetic movements to generate the two sides of a simple organ, the dorsal appendage tube of the Drosophila egg chamber. The boundary forms due to a difference in Notch levels in adjacent cells. The Notch expression pattern mimics the boundary; Notch levels are high in Rhomboid cells and low in Broad cells. Notch(-) mutant clones generate an ectopic boundary: ectopic Rhomboid cells arise in Notch(+) cells adjacent to the Notch(-) mutant cells but not further away from the clonal border. Pangolin, a component of the Wingless pathway, is required for Broad expression and for rhomboid repression. We further show that Broad represses rhomboid cell autonomously. Our data provide a foundation for understanding how a single row of Rhomboid cells arises adjacent to the Broad cells in the dorsal appendage primordia. Generating a boundary by the Notch pathway might constitute an evolutionarily conserved first step during organ formation in many tissues.     

Functional conservation of the wingless-engrailed interaction as shown by a widely applicable baculovirus misexpression system

BACKGROUND: The expression patterns of the segment polarity genes wingless and engrailed are conserved during segmentation in a variety of arthropods, suggesting that the regulatory interactions between these two genes are also evolutionarily conserved. Hypotheses derived from such comparisons of gene expression patterns are difficult to test experimentally as genetic manipulation is currently possible for only a few model organisms. RESULTS: We have developed a system, using recombinant baculoviruses, that can be applied to a wide variety of organisms to study the effects of ectopic expression of genes. As a first step, we studied the range and type of infection of several reporter viruses in the embryos of two arthropod and one vertebrate species. Using this system to express wingless, we were able to induce expression of engrailed in the anterior half of each parasegment in embryos of the fruit fly Drosophila melanogaster. Virus-mediated wingless expression also caused ectopic naked ventral cuticle formation in wild-type Drosophila larvae. In the flour beetle, Tribolium castaneum, ectopic wingless also induced engrailed expression. As in Drosophila, this expression was only detectable in the anterior half of the parasegment. CONCLUSIONS: The functional interaction between wingless and engrailed, and the establishment of cells competent to express engrailed, appears to be conserved between Drosophila and Tribolium. The data on the establishment of an engrailed-competent domain also support the idea that prepatterning by pair-rule genes is conserved between these two insects. The recombinant baculovirus technology reported here may help answer other long-standing comparative evolutionary questions.     

Secretion and localized transcription suggest a role in positional signaling for products of the segmentation gene hedgehog.

The segment polarity genes engrailed and wingless are expressed in neighboring stripes of cells on opposite sides of the Drosophila parasegment boundary. Each gene is mutually required for maintenance of the other's expression; continued expression of both also requires several other segment polarity genes. We show here that one such gene, hedgehog, encodes a protein targeted to the secretory pathway and is expressed coincidently with engrailed in embryos and in imaginal discs; maintenance of the hedgehog expression pattern is itself dependent upon other segment polarity genes including engrailed and wingless. Expression of hedgehog thus functions in, and is sensitive to, positional signaling. These properties are consistent with the non-cell autonomous requirement for hedgehog in cuticular patterning and in maintenance of wingless expression.