Role of Knot (Kn) in Wing Patterning in Drosophila

We have undertaken a genetic analysis of new strong alleles of knot (kn). The original kn(1) mutation causes an alteration of wing patterning similar to that associated with mutations of fused (fu), an apparent fusion of veins 3 and 4 in the wing. However, unlike fu, strong kn mutations do not affect embryonic segmentation and indicate that kn is not a component of a general Hh (Hedgehog)-signaling pathway. Instead we find that kn has a specific role in those cells of the wing imaginal disc that are subject to ptc-mediated Hh-signaling. Our results suggest a model for patterning the medial portion of the Drosophila wing, whereby the separation of veins 3 and 4 is maintained by kn activation in the intervening region in response to Hh-signaling across the adjacent anterior-posterior compartment boundary.     

Vein-positioning in the Drosophila wing in response to Hh; new roles of Notch signaling

The Drosophila wing is a classical model for studying the generation of developmental patterns. Previous studies have suggested that vein primordia form at boundaries between discrete sectors of gene expression along the antero-posterior (A/P) axis in the larval wing imaginal disc. Observation that the vein marker rhomboid (rho) is expressed at the centre of wider vein-competent domains led to propose that narrow vein primordia form first, and produce secondary short-range signals activating provein genes in neighbouring cells (see Curr. Opin. Genet. Dev. 10 (2000) 393). Here, we examined how the central L3 and L4 veins are positioned relative to the limits of expression of Collier (Col), a dose-dependent Hedgehog (Hh) target activated in the wing A/P organiser. We found that rho expression is first activated in broad domains adjacent to Col-expressing cells and secondarily restricted to the centre of these domains. This restriction which depends upon Notch (N) signaling sets the L3 and L4 vein primordia off the boundaries of Col expression. N activity is also required to fix the anterior limit of Col expression by locally antagonising Hh activation, thus precisely positioning the L3 vein primordium relative to the A/P compartment boundary. Experiments using Nts mutants further indicated that these two activities of N could be temporally uncoupled. Together, these observations highlight new roles of N in topologically linking the position of veins to prepattern gene expression.     

Interaction between Ataxin-2 Binding Protein 1 and Cubitus-interruptus during wing development in Drosophila

Animal growth and development is dependent on reiterative use of key signaling pathways such as Hedgehog (Hh) pathway. It is widely believed that Cubitus-interruptus (Ci) mediates all functions of Hh pathway. Here we report that CG32062, the Drosophila homologue of Ataxin-2 Binding Protein 1 (dA2BP1), functions as a cofactor of Ci to specify intervein region between L3 and L4 veins of the adult wing. Specifically, Ci-mediated transactivation of knot/collier (kn) in this region of the developing wing imaginal disc is dependent on dA2BP1 function. Protein interaction studies and chromatin-immunoprecipiation experiments suggest that Ci helps dA2BP1 to bind kn promoter, which in turn may help Ci to activate kn expression. These results suggest a mechanism by which Ci may activate targets such as kn, which do not have classical Ci/Gli-binding sites.     

Activation of knot (kn) specifies the 3-4 intervein region in the Drosophila wing

Hedgehog (Hh) plays an important role in Drosophila wing patterning by inducing expression of Dpp, which serves to organize the wing globally across the A-P axis. We show here how Hh signalling also plays a direct role in patterning the medial wing through the activation of the Hh-target gene, knot (kn). kn is expressed in Hh-responsive cells near the A-P compartment boundary, where its expression is dependent on fu, a component of Hh signalling. kn is required for the proper positioning of veins 3 and 4 and to prevent ectopic venation between them. Furthermore, the expansion anteriorly of the normal kn expression domain causes an associated anterior shift in the position of vein 3 in the resultant wing. Ectopic expression of kn elsewhere in the wing imaginal disc results in the failure to properly activate the vein initiation genes, rho and Dl. Expression of the gene encoding the EGF-receptor (EGFR), which is required for vein initiation and subsequent differentiation, is normally depressed in the 3-4 intervein region. This downregulation of EGFR in the medial portion of the imaginal disc is dependent on kn activity and ectopic expression of kn inactivates EGFR elsewhere in the wing primordium. We propose kn expression in Hh-responsive cells of the wing blade anlagen during the late third instar creates a zone of cells in the medial wing in which vein primordia cannot be induced. The primordia for veins 3 and 4 are laid down adjacent to the kn-imposed vein-free zone, presumably by a signalling factor (such as Vn) also synthesized in the medial region of the wing.     

Genetic variation for the positioning of wing veins in Drosophila melanogaster

SUMMARY To define the components of variation for wing shape in Drosophila in relation to what is known about the developmental control of wing patterning, we have characterized shape variation in the wings of 12 randomly chosen highly inbred lines. Despite large differences in wing size between males and females, and between flies reared at 18°C or 25°C, wing shape is remarkably unaffected by these variables and is highly line specific. The shape of each intervein region of the wing appears to be independently regulated at the genetic level, consistent with the role of secreted growth factors in establishing the locations of wing veins. Sex and temperature were found to have different effects on cell number in two intervein regions, with the result that wing shape is to a large extent independent of cell density. Dietary cholesterol was also shown to affect the breadth of the central intervein region, consistent with an effect on the strength of Hedgehog signaling during wing development. We conclude that wing shape is under tighter genetic control than wing size, and hypothesize that this control is achieved in large part by gene activity at the level of wing vein determination and differentiation.     

A directed mutagenesis screen in Drosophila melanogaster reveals new mutants that influence hedgehog signaling

The Hedgehog signaling pathway has been recognized as essential for patterning processes in development of metazoan animal species. The signaling pathway is, however, not entirely understood. To start to address this problem, we set out to isolate new mutations that influence Hedgehog signaling. We performed a mutagenesis screen for mutations that dominantly suppress Hedgehog overexpression phenotypes in the Drosophila melanogaster wing. We isolated four mutations that influence Hedgehog signaling. These were analyzed in the amenable wing system using genetic and molecular techniques. One of these four mutations affects the stability of the Hedgehog expression domain boundary, also known as the organizer in the developing wing. Another mutation affects a possible Hedgehog autoregulation mechanism, which stabilizes the same boundary.     

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.     

Echinoid mutants exhibit neurogenic phenotypes and show synergistic interactions with the Notch signaling pathway

During neurogenesis in Drosophila, groups of ectodermal cells are endowed with the capacity to become neuronal precursors. The Notch signaling pathway is required to limit the neuronal potential to a single cell within each group. Loss of genes of the Notch signaling pathway results in a neurogenic phenotype: hyperplasia of the nervous system accompanied by a parallel loss of epidermis. Echinoid (Ed), a cell membrane associated Immunoglobulin C2-type protein, has previously been shown to be a negative regulator of the EGFR pathway during eye and wing vein development. Using in situ hybridization and antibody staining of whole-mount embryos, we show that Ed has a dynamic expression pattern during embryogenesis. Embryonic lethal alleles of ed reveal a role of Ed in restricting neurogenic potential during embryonic neurogenesis, and result in a phenotype similar to that of loss-of-function mutations of Notch signaling pathway genes. In this process Ed interacts closely with the Notch signaling pathway. Loss of ed suppresses the loss of neuronal elements caused by ectopic activation of the Notch signaling pathway. Using a temperature-sensitive allele of ed we show, furthermore, that Ed is required to suppress sensory bristles and for proper wing vein specification during adult development. In these processes also, ed acts in close concert with genes of the Notch signaling pathway. Thus the extra wing vein phenotype of ed is enhanced upon reduction of Delta (Dl) or Enhancer of split [E(spl)] proteins. Overexpression of the membrane-tethered extracellular region of Ed results in a dominant-negative phenotype. This phenotype is suppressed by overexpression of E(spl)m7 and enhanced by overexpression of Dl. Our work establishes a role of Ed during embryonic nervous system development, as well as adult sensory bristle specification and shows that Ed interacts synergistically with the Notch signaling pathway.     

Multiple signaling pathways and a selector protein sequentially regulate Drosophila wing development

Drosophila wing development is a useful model to study organogenesis, which requires the input of selector genes that specify the identity of various morphogenetic fields (Weatherbee, S. D. and Carroll, S. B. (1999) Cell 97, 283-286) and cell signaling molecules. In order to understand how the integration of multiple signaling pathways and selector proteins can be achieved during wing development, we studied the regulatory network that controls the expression of Serrate (Ser), a ligand for the Notch (N) signaling pathway, which is essential for the development of the Drosophila wing, as well as vertebrate limbs. Here, we show that a 794 bp cis-regulatory element located in the 3' region of the Ser gene can recapitulate the dynamic patterns of endogenous Ser expression during wing development. Using this enhancer element, we demonstrate that Apterous (Ap, a selector protein), and the Notch and Wingless (Wg) signaling pathways, can sequentially control wing development through direct regulation of Ser expression in early, mid and late third instar stages, respectively. In addition, we show that later Ser expression in the presumptive vein cells is controlled by the Egfr pathway. Thus, a cis-regulatory element is sequentially regulated by multiple signaling pathways and a selector protein during Drosophila wing development. Such a mechanism is possibly conserved in the appendage outgrowth of other arthropods and vertebrates.     

A Genetic Analysis of Deltex and Its Interaction with the Notch Locus in Drosophila Melanogaster

During Drosophila development networks of genes control the developmental pathways that specify cell fates. The Notch gene is a well characterized member of some cell fate pathways, and several other genes belonging to these same pathways have been identified because they share a neurogenic null phenotype with Notch. However, it is unlikely that the neurogenic genes represent all of the genes in these pathways. The goal of this research was to use a genetic approach to identify and characterize one of the other genes that acts with Notch to specify cell fate. Mutant alleles of genes in the same pathway should have phenotypes similar to Notch alleles and should show phenotypic interactions with Notch alleles. With this approach we identified the deltex gene as a potential cell fate gene. An extensive phenotypic characterization of loss-of-function deltex phenotypes showed abnormalities (such as thick wing veins, double bristles and extra cone cells) that suggest that deltex is involved in cell fate decision processes. Phenotypic interactions between deltex and Notch as seen in double mutants showed that Notch and deltex do not code for duplicate functions and that the two genes function together in many different developing tissues. The results of these investigations lead to the conclusion that the deltex gene functions with the Notch gene in one or more developmental pathways to specify cell fate.     

Different mechanisms initiate and maintain wingless expression in the Drosophila wing hinge

The Drosophila gene wingless encodes a secreted signalling molecule that is required for many patterning events in both embryonic and postembryonic development. In the wing wingless is expressed in a complex and dynamic pattern that is controlled by several different mechanisms. These involve the Hedgehog and Notch pathways and the nuclear proteins Pannier and U-shaped. In this report, we analyse the mechanisms that drive wingless expression in the wing hinge. We present evidence that wingless is initially activated by a secreted signal that requires the genes vestigial, rotund and nubbin. Later in development, wingless expression in the wing hinge is maintained by a different mechanism, which involves an autoregulatory loop and requires the genes homothorax and rotund. We discuss the role of wingless in patterning the wing hinge.     

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.