Development of a wingless morph in the ladybird beetle, Adalia bipunctata

SUMMARY Many taxa of winged insects have independently lost the ability to fly and often possess reduced wings. Species exhibiting natural variation in wing morphology provide opportunities to investigate the genetics and developmental processes underlying the evolution of alternative wing morphs. Although many wing dimorphic species of beetles are known, the underlying mechanisms of variation are not well understood in this insect order. Here, we examine wing development of wild type and natural wingless morphs of the two-spot ladybird beetle, Adalia bipunctata . We show that both pairs of wings are distally truncated in the wingless adults. A laboratory population of the wingless morph displays heritable variation in the degree of wing truncation, reflecting reduced growth of the larval wing discs. The coexistence of variable wingless morphs supports the idea that typical monomorphic wingless insects may be the result of a gradual evolution of wing loss. Gene expression patterns in wing discs suggest that the conserved gene network controlling wing development in wild-type Adalia is disrupted in the dorsoventral patterning pathway in the wingless morphs. Previous research on several species of ant has revealed that the anteroposterior wing patterning pathway is disrupted in wingless workers. Future investigations should confirm whether interruptions in both taxa are limited to the patterning pathways found thus far, or whether there are also shared interruption points. Nevertheless, our results highlight that diverse mechanisms of development are likely to underlie the evolution of wingless insects.     

DWnt4 and wingless elicit similar cellular responses during imaginal development

Wnt genes encode evolutionarily conserved secreted proteins that provide critical functions during development. Although Wnt proteins share highly conserved features, they also show sequence divergence, which almost certainly contributes to the variety of their signaling activities. We previously reported that DWnt4 and wingless (wg), two divergent clustered Wnt genes, can have either antagonist or distinct functions during Drosophila embryogenesis. Here we provide evidence that both genes can elicit similar cellular responses during imaginal development. Ectopic expression of DWnt4 along the anterior/posterior (A/P) boundary of imaginal discs alters morphogenesis of adult appendages. In the wing disc, DWnt4 phenocopies ectopic Wg activity by inducing notum to wing transformation, suggesting similar signaling capabilities of both molecules. In support of this, we demonstrate that DWnt4 can rescue wg loss-of-function phenotypes in the antenna and haltere and is able to substitute for Wg in wing field specification. We also show that both genes are transcribed in overlapping domains in imaginal discs, suggesting that DWnt4 may cooperate with wg during limb patterning.     

A dual role for homothorax in inhibiting wing blade development and specifying proximal wing identities in Drosophila

The Drosophila wing imaginal disc gives rise to three body parts along the proximo-distal (P-D) axis: the wing blade, the wing hinge and the mesonotum. Development of the wing blade initiates along part of the dorsal/ventral (D/V) compartment boundary and requires input from both the Notch and wingless (wg) signal transduction pathways. In the wing blade, wg activates the gene vestigial (vg), which is required for the wing blade to grow. wg is also required for hinge development, but wg does not activate vg in the hinge, raising the question of what target genes are activated by wg to generate hinge structures. Here we show that wg activates the gene homothorax (hth) in the hinge and that hth is necessary for hinge development. Further, we demonstrate that hth also limits where along the D/V compartment boundary wing blade development can initiate, thus helping to define the size and position of the wing blade within the disc epithelium. We also show that the gene teashirt (tsh), which is coexpressed with hth throughout most of wing disc development, collaborates with hth to repress vg and block wing blade development. Our results suggest that tsh and hth block wing blade development by repressing some of the activities of the Notch pathway at the D/V compartment boundary.     

The wings ofBombyx mori develop from larval discs exhibiting an early differentiated state: a preliminary report

Lepidopteran insects present a complex organization of appendages which develop by various mechanisms. In the mulberry silkworm,Bombyx mori a pair of meso- and meta-thoracic discs located on either side in the larvae gives rise to the corresponding fore- and hind-wings of the adult. These discs do not experience massive cell rearrangements during metamorphosis and display the adult wing vein pattern. We have analysed wing development inB. mori by two approaches, viz., expression of patterning genes in larval wing discs, and regulatory capacities of larval discs following explantation or perturbation. Expression of Nubbin is seen all over the presumptive wing blade domains unlike inDrosophila, where it is confined to the hinge and the wing pouch. Excision of meso- and meta-thoracic discs during the larval stages resulted in emergence of adult moths lacking the corresponding wings without any loss of thoracic tissues suggesting independent origin of wing and thoracic primordia. The expression of wingless and distal-less along the dorsal/ventral margin in wing discs correlated well with their expression profile in adultDrosophila wings. Partially excised wing discs did not showin situ regeneration or duplication suggesting their early differentiation. The presence of adult wing vein patterns discernible in larval wing discs and the patterns of marker gene expression as well as the inability of these discs to regulate growth suggested that wing differentiation is achieved early inB. mori. The timings of morphogenetic events are different and the wing discs behave like presumptive wing buds opening out as wing blades inB. mori unlike evagination of only the pouch region as wing blades seen inDrosophila.     

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.     

Roles for scalloped and vestigial in regulating cell affinity and interactions between the wing blade and the wing hinge

The scalloped and vestigial genes are both required for the formation of the Drosophila wing, and recent studies have indicated that they can function as a heterodimeric complex to regulate the expression of downstream target genes. We have analyzed the consequences of complete loss of scalloped function, ectopic expression of scalloped, and ectopic expression of vestigial on the development of the Drosophila wing imaginal disc. Clones of cells mutant for a strong allele of scalloped fail to proliferate within the wing pouch, but grow normally in the wing hinge and notum. Cells overexpressing scalloped fail to proliferate in both notal and wing-blade regions of the disc, and this overexpression induces apoptotic cell death. Clones of cells overexpressing vestigial grow smaller or larger than control clones, depending upon their distance from the dorsal-ventral compartment boundary. These studies highlight the importance of correct scalloped and vestigial expression levels to normal wing development. Our studies of vestigial-overexpressing clones also reveal two further aspects of wing development. First, in the hinge region vestigial exerts both a local inhibition and a long-range induction of wingless expression. These and other observations imply that vestigial-expressing cells in the wing blade organize the development of surrounding wing-hinge cells. Second, clones of cells overexpressing vestigial exhibit altered cell affinities. Our analysis of these clones, together with studies of scalloped mutant clones, implies that scalloped- and vestigial-dependent cell adhesion contributes to separation of the wing blade from the wing hinge and to a gradient of cell affinities along the dorsal-ventral axis of the wing.     

Mutual repression between msh and Iro-C is an essential component of the boundary between body wall and wing in Drosophila

During development, the imaginal wing disc of Drosophila is subdivided into territories separated by developmental boundaries. The best characterized boundaries delimit compartments defined by cell-lineage restrictions. Here, we analyze the formation of a boundary that does not rely on such restrictions, namely, that which separates the notum (body wall) and the wing hinge (appendage). It is known that the homeobox genes of the Iroquois complex (Iro-C) define the notum territory and that the distal limit of the Iro-C expression domain demarks the boundary between the notum and the wing hinge. However, it is unclear how this boundary is established and maintained. We now find that msh, a homeobox gene of the Msx family, is strongly expressed in the territory of the hinge contiguous to the Iro-C domain. Loss- and gain-of-function analyses show that msh maintains Iro-C repressed in the hinge, while Iro-C prevents high level expression of msh in the notum. Thus, a mutual repression between msh and Iro-C is essential to set the limit between the contiguous domains of expression of these genes and therefore to establish and/or maintain the boundary between body wall and wing. In addition, we find that msh is necessary for proper growth of the hinge territory and the differentiation of hinge structures. msh also participates in the patterning of the notum, where it is expressed at low levels.     

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.     

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.     

Correlations between spatiotemporal changes in gene expression and apoptosis underlie wing polyphenism in the ant Pheidole morrisi

Wing polyphenism, which is the ability of a single genome to produce winged and wingless castes in a colony in response to environmental cues, evolved just once and is a universal feature of ants. The gene network underlying wing polyphenism, however, is conserved in the winged castes of different ant species, but is interrupted at different points in the network in the wingless castes of these species. We previously constructed a mathematical model, which predicts that a key gene brinker (brk) mediates the development and evolution of these different "interruption points" in wingless castes of different ant species. According to this model, brk is upregulated throughout the vestigial wing discs of wingless ant castes to reduce growth and induce apoptosis. Here, we tested these predictions by examining the expression of brk, as well as three other genes up- and downstream of brk-decapentaplegic (dpp), spalt (sal), and engrailed (en)-in the winged reproductive and wingless soldier castes in the ant Pheidole morrisi. We show that expression of these genes is conserved in the wing disc of winged castes. Surprisingly, however, we found that brk expression is absent throughout development of the vestigial soldier forewing disc. This absence is correlated with abnormal growth of the soldier forewing disc as revealed by En expression and morphometric analyses. We also discovered that dpp and sal expression change dynamically during the transition from larval-to-prepupal development, and is spatiotemporally correlated with the induction of apoptosis in soldier forewing disc. Our results suggest that, contrary to our predictions, brk may not be a key gene in the network for suppressing wings in soldiers, and its absence may function to disrupt the normal growth of the soldier forewing disc. Furthermore, the dynamic changes in network interruptions we discovered may be important for the induction of apoptosis, and may be a general feature of gene networks that underlie polyphenism.     

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.     

The dynamics of developmental system drift in the gene network underlying wing polyphenism in ants: a mathematical model

SUMMARY Understanding the complex interaction between genotype and phenotype is a major challenge of Evolutionary Developmental Biology. One important facet of this complex interaction has been called "Developmental System Drift" (DSD). DSD occurs when a similar phenotype, which is homologous across a group of related species, is produced by different genes or gene expression patterns in each of these related species. We constructed a mathematical model to explore the developmental and evolutionary dynamics of DSD in the gene network underlying wing polyphenism in ants. Wing polyphenism in ants is the ability of an embryo to develop into a winged queen or a wingless worker in response to an environmental cue. Although wing polyphenism is homologous across all ants, the gene network that underlies wing polyphenism has evolved. In winged ant castes, our simulations reproduced the conserved gene expression patterns observed in the network that controls wing development in holometabolous insects. In wingless ant castes, we simulated the suppression of wings by interrupting (up- or downregulating) the expression of genes in the network. Our simulations uncovered the existence of four groups of genes that have similar effects on target gene expression and growth. Although each group is comprised of genes occupying different positions in the network, their interruption produces vestigial discs that are similar in size and shape. The implications of our results for understanding the origin, evolution, and dissociation of the gene network underlying wing polyphenism in ants are discussed.     

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 Drosophila gene zfh2 is required to establish proximal-distal domains in the wing disc

Three main events characterize the development of the proximal-distal axis of the Drosophila wing disc: first, generation of nested circular domains defined by different combinations of gene expression; second, activation of wingless (wg) gene expression in a ring of cells; and third, an increase of cell number in each domain in response to Wg. The mechanisms by which these domains of gene expression are established and maintained are unknown. We have analyzed the role of the gene zinc finger homeodomain 2 (zfh2). We report that in discs lacking zfh2 the limits of the expression domains of the genes tsh, nub, rn, dve and nab coincide, and expression of wg in the wing hinge, is lost. We show that zfh2 expression is delimited distally by Vg, Nub and Dpp signalling, and proximally by Tsh and Dpp. Distal repression of zfh2 permits activation of nab in the wing blade and wg in the wing hinge. We suggest that the proximal-most wing fate, the hinge, is specified first and that later repression of zfh2 permits specification of the distal-most fate, the wing blade. We propose that proximal-distal axis development is achieved by a combination of two strategies: on one hand a process involving proximal to distal specification, with the wing hinge specified first followed later by the distal wing blade; on the other hand, early specification of the proximal-distal domains by different combinations of gene expression. The results we present here indicate that Zfh2 plays a critical role in both processes.