Wing Development Research Articles

The Hox gene Antennapedia is essential for wing development in insects.

A long-standing view in the field of evo-devo is that insect forewings develop without any Hox gene input. The Hox gene Antennapedia (Antp), despite being expressed in the thoracic segments of insects, has no effect on wing development. This view has been obtained from studies in two main model species: Drosophila and Tribolium. Here, we show that partial loss of function of Antp resulted in reduced and malformed adult wings in Bombyx, Drosophila and Tribolium. Antp mediates wing growth in Bombyx by directly regulating the ecdysteriod biosynthesis enzyme gene (shade) in the wing tissue, which leads to local production of the growth hormone 20-hydroxyecdysone. Additional targets of Antp are wing cuticular protein genes CPG24, CPH28 and CPG9, which are essential for wing development. We propose, therefore, that insect wing development occurs in an Antp-dependent manner. This article has an associated 'The people behind the papers' interview.

Post-fledging behavioral ecology of migratory songbirds: how do fledgling activity rates vary across species?

AbstractDespite a large literature on the ontogeny of animal behavior, our understanding of behavioral development in young songbirds remains relatively poor. This is particularly true for the post-fledging period, where it is often difficult to follow and observe small, freely moving fledglings for extended periods of time. Fortunately, technological advances have enabled more robust examinations of songbird post-fledging behaviors. To provide greater insight into fledgling developmental and behavioral ecology, we used automated radio-telemetry systems to document post-fledging activity rates of juveniles across 9 songbird species of east-central Illinois, USA. Specifically, we sought to examine differences in the ontogeny of activity rates across species, and compare activity rates to intrinsic (e.g. morphological traits, age) and extrinsic (e.g. predation risk, brood size) factors that have the potential to influence fledgling behavior. While post-fledging activity rates differed by species, our findings show remarkable similarities in the ontogeny of fledgling activity across the avian community; all species exhibited a steady increase in activity rates with post-fledging age, with rates leveling off roughly 3 to 4 weeks post-fledging. These general patterns mirrored rates of post-fledging mortality (primarily due to predation) across the period, including the presence or absence of a post-fledging bottleneck, in which species with bottlenecks exhibit slight declines in activity rates during the first few days out of the nest. Alongside fledgling age and mortality risk, our findings also suggest that juvenile wing development and brood size at fledging mediate fledgling activity rates within and across species.

The people behind the papers - Chunyan Fang and Xiaoling Tong.

Hox genes play a key role in determining body plan, but previous research indicated that forewing development occurs independently of Antennapedia, the Hox gene expressed in the thoracic region. Now, a new paper in Development describes an essential role for Antennapedia in wing development of silkworm, Drosophila and Tribolium. We caught up with first author, Chunyan Fang, and corresponding author, Xiaoling Tong, a group leader at the State Key Laboratory of Silkworm Genome Biology at Southwest University in China, to find out more about their research.

Broad Complex and Wing Development in Cockroaches

Broad Complex and Wing Development in Cockroaches

Use of FLP/FRT System to Screen for Notch Signaling Regulators in the Drosophila Wing.

Mutations of genes encoding key components of the Notch signaling pathways often result in lethality at early developmental stages, making it difficult to decipher how they regulate the formation of specific cell types or organs. Mosaic analysis using the FLP/FRT system allows investigating the roles of essential genes during wing development in Drosophila melanogaster. This chapter describes the practical methods to isolate Notch signaling regulators by somatic mosaic screen. The fly stocks, cross schemes, and screen parameters are summarized. We also explain how to validate the roles of potential Notch signaling regulators.

Retinoic acid influences the timing and scaling of avian wing development.

SummaryA fundamental question in biology is how embryonic development is timed between different species. To address this problem, we compared wing development in the quail and the larger chick. We reveal that pattern formation is faster in the quail as determined by the earlier activation of 5′Hox genes, termination of developmental organizers (Shh and Fgf8), and the laying down of the skeleton (Sox9). Using interspecies tissue grafts, we show that developmental timing can be reset during a critical window of retinoic acid signaling. Accordingly, extending the duration of retinoic acid signaling switches developmental timing between the quail and the chick and the chick and the larger turkey. However, the incremental growth rate is comparable between all three species, suggesting that the pace of development primarily governs differences in the expansion of the skeletal pattern. The widespread distribution of retinoic acid could coordinate developmental timing throughout the embryo.

Transcriptome Analysis Reveals the Role of Zelda in the Regulation of Embryonic and Wing Development of Tribolium Castaneum

Transcriptome Analysis Reveals the Role of Zelda in the Regulation of Embryonic and Wing Development of Tribolium Castaneum

Out from under the wing: reconceptualizing the insect wing gene regulatory network as a versatile, general module for body-wall lobes in arthropods.

Body plan evolution often occurs through the differentiation of serially homologous body parts, particularly in the evolution of arthropod body plans. Recently, homeotic transformations resulting from experimental manipulation of gene expression, along with comparative data on the expression and function of genes in the wing regulatory network, have provided a new perspective on an old question in insect evolution: how did the insect wing evolve? We investigated the metamorphic roles of a suite of 10 wing- and body-wall-related genes in a hemimetabolous insect, Oncopeltus fasciatus. Our results indicate that genes involved in wing development in O. fasciatus play similar roles in the development of adult body-wall flattened cuticular evaginations. We found extensive functional similarity between the development of wings and other bilayered evaginations of the body wall. Overall, our results support the existence of a versatile development module for building bilayered cuticular epithelial structures that pre-dates the evolutionary origin of wings. We explore the consequences of reconceptualizing the canonical wing-patterning network as a bilayered body-wall patterning network, including consequences for long-standing debates about wing homology, the origin of wings and the origin of novel bilayered body-wall structures. We conclude by presenting three testable predictions that result from this reconceptualization.

PVHL-mediated SMAD3 degradation suppresses TGF-β signaling.

Transforming growth factor β (TGF-β) signaling plays a fundamental role in metazoan development and tissue homeostasis. However, the molecular mechanisms concerning the ubiquitin-related dynamic regulation of TGF-β signaling are not thoroughly understood. Using a combination of proteomics and an siRNA screen, we identify pVHL as an E3 ligase for SMAD3 ubiquitination. We show that pVHL directly interacts with conserved lysine and proline residues in the MH2 domain of SMAD3, triggering degradation. As a result, the level of pVHL expression negatively correlates with the expression and activity of SMAD3 in cells, Drosophila wing, and patient tissues. In Drosophila, loss of pVHL leads to the up-regulation of TGF-β targets visible in a downward wing blade phenotype, which is rescued by inhibition of SMAD activity. Drosophila pVHL expression exhibited ectopic veinlets and reduced wing growth in a similar manner as upon loss of TGF-β/SMAD signaling. Thus, our study demonstrates a conserved role of pVHL in the regulation of TGF-β/SMAD3 signaling in human cells and Drosophila wing development.

Arf6 is necessary for senseless expression in response to wingless signalling during Drosophila wing development.

ABSTRACTWnt signalling is a core pathway involved in a wide range of developmental processes throughout the metazoa. In vitro studies have suggested that the small GTP binding protein Arf6 regulates upstream steps of Wnt transduction, by promoting the phosphorylation of the Wnt co-receptor, LRP6, and the release of β-catenin from the adherens junctions. To assess the relevance of these previous findings in vivo, we analysed the consequence of the absence of Arf6 activity on Drosophila wing patterning, a developmental model of Wnt/Wingless signalling. We observed a dominant loss of wing margin bristles and Senseless expression in Arf6 mutant flies, phenotypes characteristic of a defect in high level Wingless signalling. In contrast to previous findings, we show that Arf6 is required downstream of Armadillo/β-catenin stabilisation in Wingless signal transduction. Our data suggest that Arf6 modulates the activity of a downstream nuclear regulator of Pangolin activity in order to control the induction of high level Wingless signalling. Our findings represent a novel regulatory role for Arf6 in Wingless signalling.

Selection of Reference Genes for RT-qPCR Analysis of Wing Dimorphism in English Grain Aphid, Sitobion avenae (Hemiptera: Aphididae).

The English grain aphid, Sitobion avenae (Fabricius), exhibits classic and dramatic phenotypic plasticity in wing development. Both genetic and environmental inputs contribute to the wing polyphenism in aphids, an extreme form of phenotypic plasticity in which a single genotype produces discrete winged and wingless morphs. Validated reference genes are needed to accurately normalize temporal and spatial variation in gene expression estimates by RT-qPCR. In this research, the stability of 11 candidate reference genes selected from S. avenae transcriptomes was evaluated under an array of abiotic and biotic conditions relevant to wing development. RefFinder, a comprehensive software integrating rankings from delta Ct, BestKeeper, NormFinder, and geNorm, offered a series of reference genes for every experimental condition. Overall, helicase (HEL) and ubiquitin ribosomal protein S27A fusion protein (RpS27) are suited for most of the conditions examined in this study, although exceptions do exist. Specifically, NADH dehydrogenase (Ap-NADH) and 28S ribosomal RNA (28S) are recommended for insecticide and antibiotic treatments, while ribosomal RNA L14 (RPL14) and 18S ribosomal RNA (18S) are selected for density treatment, respectively. This study provides a suite of reference genes to investigate the wing polyphenism in S. avenae, and is important for application of RT-qPCR in future experiments of novel tactics to control aphids.

Wing hearts in four-winged Ultrabithorax-mutant flies-the role of Hox genes in wing heart specification.

Wings are probably the most advanced evolutionary novelty in insects. In the fruit fly Drosophila melanogaster, proper development of wings requires the activity of so-called wing hearts located in the scutellum of the thorax. Immediately after the imaginal ecdysis, these accessory circulatory organs remove hemolymph and apoptotic epidermal cells from the premature wings through their pumping action. This clearing process is essential for the formation of functional wing blades. Mutant flies that lack intact wing hearts are flightless and display malformed wings. The embryonic wing heart progenitors originate from two adjacent parasegments corresponding to the later second and third thoracic segments. However, adult dipterian flies harbor only one pair of wings and only one pair of associated wing hearts in the second thoracic segment. Here we show that the specification of WHPs depends on the regulatory activity of the Hox gene Ultrabithorax. Furthermore, we analyzed the development of wing hearts in the famous four-winged Ultrabithorax (Ubx) mutant, which was first discovered by Ed Lewis in the 1970s. In these flies, the third thoracic segment is homeotically transformed into a second thoracic segment resulting in a second pair of wings instead of the club-shaped halteres. We show that a second pair of functional wing hearts is formed in the transformed third thoracic segment and that all wing hearts originate from the wild-type population of wing heart progenitor cells.

Early cellular development induced by ecdysteroid in sex-specific wing degeneration of the wingless female winter moth.

The winter moth, Nyssiodes lefuarius, exhibits striking sexual dimorphism in wing form; males have functional wings of normal size, whereas females lack wings. We previously found that the steroid hormone 20-hydroxyecdysone (20E) triggered massive programmed cell death (PCD) only in the female pupal wing epithelium; however, when and how early sexual trait development of the pupal wings is initiated during pupal-adult metamorphosis remains obscure. To clarify the detailed morphological changes and mechanisms underlying early sexual trait development and cell death, we examined the effects of 20E on early ultrastructural and histological changes in the pupal wing epithelium of both sexes. Before the onset of adult differentiation, no morphological differences were observed in the epithelial cells of both sexes at an ultrastructural level. When 5.4µg of 20E was injected into pupae of both sexes at 15days after the onset of pupation, retraction of the wing epithelium from the pupal cuticle was initiated at day 2 after 20E injection in both sexes. Although overt degeneration of wing tissue was not still obvious, apoptotic body-like structures and auto-phagosomes were visible at day 3 after 20E injection in females, whereas development of scale precursor cells started on day 4 after injection in males. Our results suggest that (1) the injection of 20E induced sexually dimorphic changes in the pattern of organelle distribution in wing epithelial cells, and (2) abnormally shaped mitochondria in the cytoplasm of the female wing epithelium might be involved in the PCD that occurs during wing tissue degeneration.

Nanocarrier-delivered dsRNA suppresses wing development of green peach aphids.

RNA interference (RNAi) has developed rapidly as a potential "green" pest management strategy. At present, most studies have focused on the screening of aphid lethal genes, whereas only a few studies have been conducted on wing development, which is crucial for aphid migration and plant-virus dissemination. Here, the Myzus persicae genes vestigial (vg) and Ultrabithorax (Ubx) related to wing development, were cloned. These two genes were expressed in various tissues of 3rd-instar winged aphids. The mRNA level of vg was high in 3rd-instar nymphs, whereas the expression level of Ubx was high in adults. The nanocarrier-mediated delivery system delivered double-stranded RNAs for aphid RNAi using topical and root applications. The expression levels of vg and Ubx were downregulated by 44.0% and 36.5%, respectively, using the topical application. The simultaneous RNAi of the two target genes caused 63.3% and 32.2% wing aberration rates using topical and root applications, respectively. The current study provided a promising method for controlling aphid migration to alleviate the spread of insect transmitted plant diseases.

The Ubiquitin Conjugating Enzyme UbcD1 is Required for Notch Signaling Activation During Drosophila Wing Development

Notch signaling pathway plays crucial roles in animal development. Protein ubiquitination contributes to Notch signaling regulation by governing the stability and activity of major signaling components. Studies in Drosophila have identified multiple ubiquitin ligases and deubiquitinating enzymes that modify Notch ligand and receptor proteins. The fate of ubiquitinated substrates depend on topologies of the attached ubiquitin chains, which are determined by the ubiquitin conjugating enzymes (E2 enzymes). However, which E2 enzymes participate in Notch signal transduction remain elusive. Here, we report that the E2 enzyme UbcD1 is required for Notch signaling activation during Drosophila wing development. Mutations of UbcD1 lead to marginal nicks in the adult wing and reduction of Notch signaling targets expression in the wing imaginal disc. Genetic analysis reveal that UbcD1 functions in the signaling receiving cells prior to cleavage of the Notch protein. We provide further evidence suggesting that UbcD1 is likely involved in endocytic trafficking of Notch protein. Our results demonstrate that UbcD1 positively regulates Notch signaling and thus reveal a novel role of UbcD1 in development.

Dynein Heavy Chain 64C Differentially Regulates Cell Survival and Proliferation of Wingless-Producing Cells in Drosophila melanogaster.

Dynein is a multi-subunit motor protein that moves toward the minus-end of microtubules, and plays important roles in fly development. We identified Dhc64Cm115, a new mutant allele of the fly Dynein heavy chain 64C (Dhc64C) gene whose heterozygotes survive against lethality induced by overexpression of Sol narae (Sona). Sona is a secreted metalloprotease that positively regulates Wingless (Wg) signaling, and promotes cell survival and proliferation. Knockdown of Dhc64C in fly wings induced extensive cell death accompanied by widespread and disorganized expression of Wg. The disrupted pattern of the Wg protein was due to cell death of the Wg-producing cells at the DV midline and overproliferation of the Wg-producing cells at the hinge in disorganized ways. Coexpression of Dhc64C RNAi and p35 resulted in no cell death and normal pattern of Wg, demonstrating that cell death is responsible for all phenotypes induced by Dhc64C RNAi expression. The effect of Dhc64C on Wg-producing cells was unique among components of Dynein and other microtubule motors. We propose that Dhc64C differentially regulates survival of Wg-producing cells, which is essential for maintaining normal expression pattern of Wg for wing development.

Genome-wide analysis of long non-coding RNAs and their association with wing development in Aphis citricidus (Hemiptera: Aphididae)

Long non-coding RNAs (lncRNAs) play critical roles in the various physiological processes of insects. The wing is a successful adaptation allowing insects to escape from unfavorable environments, while information on lncRNAs related to wing development is limited. In this study, we constructed 12 libraries from two RNA-seq comparisons: 4th instar winged nymphs versus winged adults and 4th instar wingless nymphs versus wingless adults in the brown citrus aphid Aphis citricidus, to identify the wing development-associated lncRNAs. A total of 2914 lncRNAs were identified and 50 lncRNAs were differentially expressed during the 4th instar winged nymphs to winged adults transition, and 28 lncRNAs changed during the 4th instar wingless nymphs to wingless adults transition. The differentially expressed lncRNAs were grouped into six clusters according to the expression patterns in the combined two-winged morphs. lncRNA Ac_lnc54106.1 was up-regulated during 4th instar winged nymphs to winged adults transition, but a lack of change during the 4th instar wingless nymphs to wingless adults transition implied a critical role in the specific regulation of wing development. RNA interference of Ac_lnc54106.1 resulted in malformed wings. Targets prediction, expression patterns, and RNAi assay results showed that Ac_lnc54106.1 may target the PiggyBac transposable element-derived protein 4 (PGBD4) gene, decrease expression of the canonical wing development-related genes, and finally regulate wing development. The systematic identification of lncRNAs in an aphid increases our understanding of how non-coding RNA mediates the wing plasticity of insects.

Genome-wide phenotypic RNAi screen in the Drosophila wing: phenotypic description of functional classes.

The Drosophila genome contains approximately 14,000 protein-coding genes encoding all the necessary information to sustain cellular physiology, tissue organization, organism development, and behavior. In this manuscript, we describe in some detail the phenotypes in the adult fly wing generated after knockdown of approximately 80% of Drosophila genes. We combined this phenotypic description with a comprehensive molecular classification of the Drosophila proteins into classes that summarize the main expected or known biochemical/functional aspect of each protein. This information, combined with mRNA expression levels and in situ expression patterns, provides a simplified atlas of the Drosophila genome, from housekeeping proteins to the components of the signaling pathways directing wing development, that might help to further understand the contribution of each gene group to wing formation.

Pegasus, a small extracellular peptide enhancing short-range diffusion of Wingless

Small Open Reading Frames (smORFs) coding for peptides of less than 100 amino-acids are an enigmatic and pervasive gene class, found in the tens of thousands in metazoan genomes. Here we reveal a short 80 amino-acid peptide (Pegasus) which enhances Wingless/Wnt1 protein short-range diffusion and signalling. During Drosophila wing development, Wingless has sequential functions, including late induction of proneural gene expression and wing margin development. Pegasus mutants produce wing margin defects and proneural expression loss similar to those of Wingless. Pegasus is secreted, and co-localizes and co-immunoprecipitates with Wingless, suggesting their physical interaction. Finally, measurements of fixed and in-vivo Wingless gradients support that Pegasus increases Wingless diffusion in order to enhance its signalling. Our results unveil a new element in Wingless signalling and clarify the patterning role of Wingless diffusion, while corroborating the link between small open reading frame peptides, and regulation of known proteins with membrane-related functions.

Lepidoptera demonstrate the relevance of Murray\u2019s Law to circulatory systems with tidal flow

BackgroundMurray’s Law, which describes the branching architecture of bifurcating tubes, predicts the morphology of vessels in many amniotes and plants. Here, we use insects to explore the universality of Murray’s Law and to evaluate its predictive power for the wing venation of Lepidoptera, one of the most diverse insect orders. Lepidoptera are particularly relevant to the universality of Murray’s Law because their wing veins have tidal, or oscillatory, flow of air and hemolymph. We examined over one thousand wings representing 667 species of Lepidoptera.ResultsWe found that veins with a diameter above approximately 50 microns conform to Murray’s Law, with veins below 50 microns in diameter becoming less and less likely to conform to Murray’s Law as they narrow. The minute veins that are most likely to deviate from Murray’s Law are also the most likely to have atrophied, which prevents efficient fluid transport regardless of branching architecture. However, the veins of many taxa continue to branch distally to the areas where they atrophied, and these too conform to Murray’s Law at larger diameters (e.g., Sesiidae).ConclusionsThis finding suggests that conformity to Murray’s Law in larger taxa may reflect requirements for structural support as much as fluid transport, or may indicate that selective pressures for fluid transport are stronger during the pupal stage—during wing development prior to vein atrophy—than the adult stage. Our results increase the taxonomic scope of Murray’s Law and provide greater clarity about the relevance of body size.