Pupal Wing Research Articles

Coronin-1 promotes directional cell rearrangement in Drosophila wing epithelium.

Directional cell rearrangement is a critical process underlying correct tissue deformation during morphogenesis. Although the involvement of F-actin regulation in cell rearrangement has been established, the role and regulation of actin binding proteins (ABPs) in this process are not well understood. In this study, we investigated the function of Coronin-1, a WD-repeat actin-binding protein, in controlling directional cell rearrangement in the Drosophila pupal wing. Transgenic flies expressing Coronin-1-EGFP were generated using CRISPR-Cas9. We observed that Coronin-1 localizes at the reconnecting junction during cell rearrangement, which is dependent on actin interacting protein 1 (AIP1) and cofilin, actin disassemblers and known regulators of wing cell rearrangement. Loss of Coronin-1 function reduces cell rearrangement directionality and hexagonal cell fraction. These results suggest that Coronin-1 promotes directional cell rearrangement via its interaction with AIP1 and cofilin, highlighting the role of ABPs in the complex process of morphogenesis.Key words: morphogenesis, cell rearrangement, actin binding proteins (ABPs).

Butterfly Wing Color Pattern Modification Inducers May Act on Chitin in the Apical Extracellular Site: Implications in Morphogenic Signals for Color Pattern Determination.

Butterfly wing color patterns are modified by various treatments, such as temperature shock, injection of chemical inducers, and covering materials on pupal wing tissue. Their mechanisms of action have been enigmatic. Here, we investigated the mechanisms of color pattern modifications usingthe blue pansy butterfly Junoniaorithya. We hypothesized that these modification-inducing treatments act on the pupal cuticle or extracellular matrix (ECM). Mechanical load tests revealed that pupae treated with cold shock or chemical inducers were significantly less rigid, suggesting that these treatments made cuticle formation less efficient. A known chitin inhibitor, FB28 (fluorescent brightener 28), was discovered to efficiently induce modifications. Taking advantage of its fluorescent character, fluorescent signals from FB28 were observed in live pupae in vivo from the apical extracellular side and were concentrated at the pupal cuticle focal spots immediately above the eyespot organizing centers. It was shown that chemical modification inducers and covering materials worked additively. Taken together, various modification-inducing treatments likely act extracellularly on chitin or other polysaccharides to inhibit pupal cuticle formation or ECM function, which probably causes retardation of morphogenic signals. It is likely that an interactive ECM is required for morphogenic signals for color pattern determination to travel long distances.

Distinct mechanisms of planar polarization by the core and Fat-Dachsous planar polarity pathways in the Drosophila wing.

Planar polarity describes the coordinated polarization of cells within a tissue plane, and in animals can be determined by the "core" or Fat-Dachsous pathways. Current models for planar polarity establishment involve two components: tissue-level "global" cues that determine the overall axis of polarity and cell-level feedback-mediated cellular polarity amplification. Here, we investigate the contributions of global cues versus cellular feedback amplification in the core and Fat-Dachsous pathways during Drosophila pupal wing development. We present evidence that these pathways generate planar polarity via distinct mechanisms. Core pathway function is consistent with strong feedback capable of self-organizing cell polarity, which can then be aligned with the tissue axis via weak or transient global cues. Conversely, generation of cell polarity by the Ft-Ds pathway depends on strong global cues in the form of graded patterns of gene expression, which can then be amplified by weak feedback mechanisms.

Laser Microdissection-Mediated Isolation of Butterfly Wing Tissue for Spatial Transcriptomics.

The assignment of specific patterns of gene expression to specific cells in a complex tissue facilitates the connection between genotype and phenotype. Single-cell sequencing of whole tissues produces single-cell transcript resolution but lacks the spatial information of the derivation of each cell, whereas techniques such as multiplex FISH localize transcripts to specific cells in a tissue but require a priori information of the target transcripts to examine. Laser dissection of tissues followed by transcriptome analysis is an efficient and cost-effective technique that provides both unbiased gene expression discovery together with spatial information. Here, we detail a laser dissection protocol for total RNA extraction from butterfly larval and pupal wing tissues, without the need of paraffin embedding or the use of a microtome, that could be useful to researchers interested in the transcriptome of specific areas of the wing during development. This protocol can bypass difficulties in extracting high quality RNA from thick fixed tissues for sequencing applications.

Do field collected immature stages of Aedes mosquitoes exhibit correspondence of the life-history traits? Observations from Kolkata, India

ABSTRACT Entomological monitoring of dengue vectors Aedes aegypti (Linnaeus, 1762) and Ae. albopictus (Skuse, 1894) (Diptera: Culicidae) provides crucial information on the life-history traits (LHTs). An assessment of the LHTs of Aedes mosquitoes was accomplished using randomly collected immature stages from various larval habitats of Kolkata, India. Out of 104 larval habitats ~43% were exclusive for Ae. aegypti, ~31% were exclusive for Ae. albopictus and ~26% were common for both the species. Both female and male Ae. aegypti were bigger than the equivalents of Ae. albopictus in terms of pupal weight, adult weight and wing length. All the LHTs were significantly (P < 0.05) correlated, and the sexual dimorphism was evident for both Aedes mosquitoes. In both the species, the fecundity (ln-transformed value; y) increased with the wing length (x) [for Ae. aegypti y = 1.012x+1.023, R2 = 0.455, and for Ae. albopictus y = 1.022x+1.083, R2 = 0.154]. A correspondence of the larval development and the adult traits was evident for both the Aedes mosquitoes. Incorporation of the life-history trait data with pupal productivity in the surveillance of the dengue vectors will enable precise predictions about the population abundance and possibility of disease transmission.

Distal-less and spalt are distal organisers of pierid wing patterns

Two genes, Distal-less (Dll) and spalt (sal), are known to be involved in establishing nymphalid butterfly wing patterns. They function in several ways: in the differentiation of the eyespot’s central signalling cells, or foci; in the differentiation of the surrounding black disc; in overall scale melanisation (Dll); and in elaborating marginal patterns, such as parafocal elements. However, little is known about the functions of these genes in the development of wing patterns in other butterfly families. Here, we study the expression and function of Dll and sal in the development of spots and other melanic wing patterns of the Indian cabbage white, Pieris canidia, a pierid butterfly. In P. canidia, both Dll and Sal proteins are expressed in the scale-building cells at the wing tips, in chevron patterns along the pupal wing margins, and in areas of future scale melanisation. Additionally, Sal alone is expressed in the future black spots. CRISPR knockouts of Dll and sal showed that each gene is required for the development of melanic wing pattern elements, and repressing pteridine granule formation, in the areas where they are expressed. We conclude that both genes likely play ancestral roles in organising distal butterfly wing patterns, across pierid and nymphalid butterflies, but are unlikely to be differentiating signalling centres in pierids black spots. The genetic and developmental mechanisms that set up the location of spots and eyespots are likely distinct in each lineage.

Selective function of the PDZ domain of Dishevelled in noncanonical Wnt signalling.

ABSTRACTDishevelled is a cytoplasmic hub that transduces Wnt signals to cytoplasmic effectors, which can be broadly characterised as canonical (β-catenin dependent) and noncanonical, to specify cell fates and behaviours during development. To transduce canonical Wnt signals, Dishevelled binds to the intracellular face of Frizzled through its DEP domain and polymerises through its DIX domain to assemble dynamic signalosomes. Dishevelled also contains a PDZ domain, whose function remains controversial. Here, we use genome editing to delete the PDZ domain-encoding region from Drosophila dishevelled. Canonical Wingless signalling is entirely normal in these deletion mutants; however, they show defects in multiple contexts controlled by noncanonical Wnt signalling, such as planar polarity. We use nuclear magnetic resonance spectroscopy to identify bona fide PDZ-binding motifs at the C termini of different polarity proteins. Although deletions of these motifs proved aphenotypic in adults, we detected changes in the proximodistal distribution of the polarity protein Flamingo (also known as Starry night) in pupal wings that suggest a modulatory role of these motifs in polarity signalling. We also provide new genetic evidence that planar polarity relies on the DEP-dependent recruitment of Dishevelled to the plasma membrane by Frizzled.

Use of Fluorescence Recovery After Photobleaching (FRAP) to Measure In Vivo Dynamics of Cell Junction-Associated Polarity Proteins.

Here, we present a detailed protocol for fluorescence recovery after photobleaching (FRAP) to measure the dynamics of junctional populations of proteins in living tissue. Specifically, we describe how to perform FRAP in Drosophila pupal wings on fluorescently tagged core planar polarity proteins, which exhibit relatively slow junctional turnover. We provide a step-by-step practical guide to performing FRAP, and list a series of controls and optimizations to do before conducting a FRAP experiment. Finally, we describe how to present the FRAP data for publication.

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.

RASSF8-mediated transport of Echinoid via the exocyst promotes Drosophila wing elongation and epithelial ordering.

ABSTRACTCell-cell junctions are dynamic structures that maintain cell cohesion and shape in epithelial tissues. During development, junctions undergo extensive rearrangements to drive the epithelial remodelling required for morphogenesis. This is particularly evident during axis elongation, where neighbour exchanges, cell-cell rearrangements and oriented cell divisions lead to large-scale alterations in tissue shape. Polarised vesicle trafficking of junctional components by the exocyst complex has been proposed to promote junctional rearrangements during epithelial remodelling, but the receptors that allow exocyst docking to the target membranes remain poorly understood. Here, we show that the adherens junction component Ras Association domain family 8 (RASSF8) is required for the epithelial re-ordering that occurs during Drosophila pupal wing proximo-distal elongation. We identify the exocyst component Sec15 as a RASSF8 interactor. Loss of RASSF8 elicits cytoplasmic accumulation of Sec15 and Rab11-containing vesicles. These vesicles also contain the nectin-like homophilic adhesion molecule Echinoid, the depletion of which phenocopies the wing elongation and epithelial packing defects observed in RASSF8 mutants. Thus, our results suggest that RASSF8 promotes exocyst-dependent docking of Echinoid-containing vesicles during morphogenesis.

Comparative transcriptome analysis at the onset of speciation in a mimetic butterfly-The Ithomiini Melinaea marsaeus.

Ecological speciation entails divergent selection on specific traits and ultimately on the developmental pathways responsible for these traits. Selection can act on gene sequences but also on regulatory regions responsible for gene expression. Mimetic butterflies are a relevant system for speciation studies because wing colour pattern (WCP) often diverges between closely related taxa and is thought to drive speciation through assortative mating and increased predation on hybrids. Here, we generate the first transcriptomic resources for a mimetic butterfly of the tribe Ithomiini, Melinaea marsaeus, to examine patterns of differential expression between two subspecies and between tissues that express traits that likely drive reproductive isolation; WCP and chemosensory genes. We sequenced whole transcriptomes of three life stages to cover a large catalogue of transcripts, and we investigated differential expression between subspecies in pupal wing discs and antennae. Eighteen known WCP genes were expressed in wing discs and 115 chemosensory genes were expressed in antennae, with a remarkable diversity of chemosensory protein genes. Many transcripts were differentially expressed between subspecies, including two WCP genes and one odorant receptor. Our results suggest that in M.marsaeus the same genes as in other mimetic butterflies are involved in traits causing reproductive isolation, and point at possible candidates for the differences in those traits between subspecies. Differential expression analyses of other developmental stages and body organs and functional studies are needed to confirm and expand these results. Our work provides key resources for comparative genomics in mimetic butterflies, and more generally in Lepidoptera.

Mechano-chemical feedback mediated competition for BMP signalling leads to pattern formation

Developmental patterning is thought to be regulated by conserved signalling pathways. Initial patterns are often broad before refining to only those cells that commit to a particular fate. However, the mechanisms by which pattern refinement takes place remain to be addressed. Using the posterior crossvein (PCV) of the Drosophila pupal wing as a model, into which bone morphogenetic protein (BMP) ligand is extracellularly transported to instruct vein patterning, we investigate how pattern refinement is regulated. We found that BMP signalling induces apical enrichment of Myosin II in developing crossvein cells to regulate apical constriction. Live imaging of cellular behaviour indicates that changes in cell shape are dynamic and transient, only being maintained in those cells that retain vein fate competence after refinement. Disrupting cell shape changes throughout the PCV inhibits pattern refinement. In contrast, disrupting cell shape in only a subset of vein cells can result in a loss of BMP signalling. We propose that mechano-chemical feedback leads to competition for the developmental signal which plays a critical role in pattern refinement.

RNA In Situ Hybridization for Detecting Gene Expression Patterns in the Abdomens and Wings of Drosophila Species.

RNA in situ hybridization (ISH) is used to visualize spatio-temporal gene expression patterns with broad applications in biology and biomedicine. Here we provide a protocol for mRNA ISH in developing pupal wings and abdomens for model and non-model Drosophila species. We describe best practices in pupal staging, tissue preparation, probe design and synthesis, imaging of gene expression patterns, and image-editing techniques. This protocol has been successfully used to investigate the roles of genes underlying the evolution of novel color patterns in non-model Drosophila species.

DIA-based proteome reveals the involvement of cuticular proteins and lipids in the wing structure construction in the silkworm

Wing discs of Bombyx mori (B. mori) are transformed into wings during metamorphosis via dramatic morphological and structural changes. Mutations in genes related to the wings cause the adults to have altered wing shapes or abnormal wing colour. At present, there are more than 20 wing mutants recorded in the silkworm. However, the key factors that influence B. mori wing development are still unclear. Here, we used the strains +Wes/+Wes and Wes/+Wes that are typical for the normal wing and shriveled wing phenotypes, respectively, to identify differentially expressed proteins by label-free data-independent acquisition (DIA). Ten enriched GO terms and 9 KEGG pathways were identified based on the 3993 proteins in the wings. Among the identified and quantified proteins, 370 differentially expressed proteins (DEPs) were detected (P-value <0.01, |log2FC| > 0.58). Mapping of the DEPs to the reference canonical pathways in KEGG showed that the top 20% of the pathways were related to fatty acid, cutin, suberin and wax biosynthesis, protein processing in endoplasmic reticulum, protein export, etc. Of the 370 DEPs, 238 were down-regulated, and 132 were up-regulated of Wes/+Wes compared with +Wes/+Wes. Numerous cuticular proteins were down-regulated, and fatty metabolism enzymes were up-regulated, in Wes/+Wes compared with +Wes/+Wes. SignificanceThe comparative analysis of proteomes suggested that cuticular proteins and fatty metabolism enzymes are the main abnormally expressed proteins in the pupal wings of Wes/+Wes, leading to curly and shrunken wings after moth transformation. Our results also identify the substances affecting the development of silkworm wings from the perspective of proteins. The information from this study is important for further research on the molecular mechanisms of wing development in lepidopteran insects, and these differentially expressed genes may be targets for Lepidoptera pest control.

Cell Dissociation from Butterfly Pupal Wing Tissues for Single-Cell RNA Sequencing

Butterflies are well known for their beautiful wings and have been great systems to understand the ecology, evolution, genetics, and development of patterning and coloration. These color patterns are mosaics on the wing created by the tiling of individual units called scales, which develop from single cells. Traditionally, bulk RNA sequencing (RNA-seq) has been used extensively to identify the loci involved in wing color development and pattern formation. RNA-seq provides an averaged gene expression landscape of the entire wing tissue or of small dissected wing regions under consideration. However, to understand the gene expression patterns of the units of color, which are the scales, and to identify different scale cell types within a wing that produce different colors and scale structures, it is necessary to study single cells. This has recently been facilitated by the advent of single-cell sequencing. Here, we provide a detailed protocol for the dissociation of cells from Bicyclus anynana pupal wings to obtain a viable single-cell suspension for downstream single-cell sequencing. We outline our experimental design and the use of fluorescence-activated cell sorting (FACS) to obtain putative scale-building and socket cells based on size. Finally, we discuss some of the current challenges of this technique in studying single-cell scale development and suggest future avenues to address these challenges.

Morphological and histological examination of short-wing formation in the winter moth Protalcis concinnata (Insecta: Lepidoptera, Geometridae).

Winter geometrid moths exhibit sexual dimorphism in wing length and female-specific flightlessness. Female-specific flightlessness in insects is an interesting phenomenon in terms of sexual dimorphism and reproductive biology. In the winter geometrid moth, Protalcis concinnata (Wileman), adult females have short wings and adult males have fully developed wings. Although the developmental process for wing reduction in Lepidoptera is well studied, little is known about the morphology and the developmental pattern of short-winged flightless morphs in Lepidoptera. To clarify the precise mechanisms and developmental processes that produce short-winged morphs, we performed morphological and histological investigations of adult and pupal wing development in the winter geometrid moth P. concinnata. Our findings showed that (a) wing development in both sexes is similar until larval-pupal metamorphosis, (b) the shape of the sexually dimorphic wings is determined by the position of the bordering lacuna (BL), (c) the BL is positioned farther inward in females than in males, and (d) after the short pupal diapause period, the female pupal wing epithelium degenerates to approximately two-thirds its original size due to cell death. We propose that this developmental pattern is a previously unrecognized process among flightless Lepidoptera.

A release from developmental bias accelerates morphological diversification in butterfly eyespots

Development can bias the independent evolution of traits sharing ontogenetic pathways, making certain evolutionary changes less likely. The eyespots commonly found on butterfly wings each have concentric rings of differing colors, and these serially repeated pattern elements have been a focus for evo-devo research. In the butterfly family Nymphalidae, eyespots have been shown to function in startling or deflecting predators and to be involved in sexual selection. Previous work on a model species of Mycalesina butterfly, Bicyclus anynana, has provided insights into the developmental control of the size and color composition of individual eyespots. Experimental evolution has also shown that the relative size of a pair of eyespots on the same wing surface is highly flexible, whereas they are resistant to diverging in color composition, presumably due to the underlying shared developmental process. This fixed color composition has been considered as a prime example of developmental bias with significant consequences for wing pattern evolution. Here, we test this proposal by surveying eyespots across the whole subtribe of Mycalesina butterflies and demonstrate that developmental bias shapes evolutionary diversification except in the genus Heteropsis which has gained independent control of eyespot color composition. Experimental manipulations of pupal wings reveal that the bias has been released through a novel regional response of the wing tissue to a conserved patterning signal. Our study demonstrates that development can bias the evolutionary independence of traits, but it also shows how bias can be released through developmental innovations, thus, allowing rapid morphological change, facilitating evolutionary diversification.

Overexpressed Gliotactin activates BMP signaling through interfering with the Tkv-Dad association.

Epithelial junctions ensure cell-cell adhesion and establish permeability barriers between cells. At the corners of epithelia, the tricellular junction (TCJ) is formed by three adjacent epithelial cells and generates a functional barrier. In Drosophila, a key TCJ protein is Gliotactin (Gli) where loss of Gli disrupts barrier formation and function. Conversely, overexpressed Gli spreads away from the TCJ and triggers apoptosis, delamination, and cell migration. Thus, Gli protein levels are tightly regulated and by two mechanisms, at the protein levels by tyrosine phosphorylation and endocytosis and at the mRNA level through microRNA-184. Regulation of Gli mRNA is mediated through a Gli-BMP-miR184 feedback loop. Excessive Gli triggers BMP signaling pathway through the activation of Tkv type-I BMP receptor and Mad. Elevated level of pMad induces micrRNA-184 expression which in turn targets the Gli 3'UTR and mRNA degradation. Gli activation of Tkv is not through its ligand Dpp but rather through the inhibition of Dad, an inhibitory-Smad. Here, we show that ectopic expression of Gli interferes with Tkv-Dad association by sequestering Dad away from Tkv. The reduced inhibitory effect of Dad on Tkv results in the increased Tkv-pMad signaling activity, and this effect is continuous through larval and pupal wing formation.

Observation of morphological abnormalities in silkworm pupae after feeding 137CsCl-supplemented diet to evaluate the effects of low dose-rate exposure

Since the Fukushima Dai-ichi Nuclear Power Plant (FDNPP) accident, morphological abnormalities in lepidopteran insects, such as shrinkage and/or aberration of wings, have been reported. Butterflies experimentally exposed to radiocesium also show such abnormalities. However, because of a lack of data on absorbed dose and dose–effect relationship, it is unclear whether these abnormalities are caused directly by radiation. We conducted a low dose-rate exposure experiment in silkworms reared from egg to fully developed larvae on a 137CsCl-supplemented artificial diet and estimated the absorbed dose to evaluate morphological abnormalities in pupal wings. We used 137CsCl at 1.3 × 103 Bq/g fresh weight to simulate 137Cs contamination around the FDNPP. Absorbed doses were estimated using a glass rod dosimeter and Monte Carlo particle transport simulation code PHITS. Average external absorbed doses were approximately 0.24 (on diet) and 0.016 mGy/day (near diet); the average internal absorbed dose was approximately 0.82 mGy/day. Pupal wing structure is sensitive to radiation exposure. However, no significant differences were observed in the wing-to-whole body ratio of pupae between the 137CsCl-exposure and control groups. These results suggest that silkworms are insensitive to low dose-rate exposure due to chronic ingestion of high 137Cs at a high concentration.

DNA methylation suppresses chitin degradation and promotes the wing development by inhibiting Bmara-mediated chitinase expression in the silkworm, Bombyx mori

BackgroundDNA methylation, as an essential epigenetic modification found in mammals and plants, has been implicated to play an important role in insect reproduction. However, the functional role and the regulatory mechanism of DNA methylation during insect organ or tissue development are far from being clear.ResultsHere, we found that DNA methylation inhibitor (5-aza-dC) treatment in newly molted pupae decreased the chitin content of pupal wing discs and adult wings and resulted in wing deformity of Bombyx mori. Transcriptome analysis revealed that the up-regulation of chitinase 10 (BmCHT10) gene might be related to the decrease of chitin content induced by 5-aza-dC treatment. Further, the luciferase activity assays demonstrated that DNA methylation suppressed the promoter activity of BmCHT10 by down-regulating the transcription factor, homeobox protein araucan (Bmara). Electrophoretic mobility shift assay, DNA pull-down and chromatin immunoprecipitation demonstrated that Bmara directly bound to the BmCHT10 promoter. Therefore, DNA methylation is involved in keeping the structural integrity of the silkworm wings from unwanted chitin degradation, as a consequence, it promotes the wing development of B. mori.ConclusionsThis study reveals that DNA methylation plays an important role in the wing development of B. mori. Our results support that the indirect transcriptional repression of a chitin degradation-related gene BmCHT10 by DNA methylation is necessary to keep the proper wing development in B. mori.