The actin cytoskeleton plays multiple roles in structural colour formation in butterfly wing scales.

Vivid structural colours in butterflies are caused by photonic nanostructures scattering light. Structural colours evolved for numerous biological signalling functions and have important technological applications. Optically, such structures are well understood, however insight into their development in vivo remains scarce. We show that actin is intimately involved in structural colour formation in butterfly wing scales. Using comparisons between iridescent (structurally coloured) and non-iridescent scales in adult and developing H. sara, we show that iridescent scales have more densely packed actin bundles leading to an increased density of reflective ridges. Super-resolution microscopy across three distantly related butterfly species reveals that actin is repeatedly re-arranged during scale development and crucially when the optical nanostructures are forming. Furthermore, actin perturbation experiments at these later developmental stages resulted in near total loss of structural colour in H. sara. Overall, this shows that actin plays a vital and direct templating role during structural colour formation in butterfly scales, providing ridge patterning mechanisms that are likely universal across lepidoptera.

The AAA-ATPase Ter94 regulates wing size in Drosophila by suppressing the Hippo pathway.

Insect wing development is a fascinating and intricate process that involves the regulation of wing size through cell proliferation and apoptosis. In this study, we find that Ter94, an AAA-ATPase, is essential for proper wing size dependently on its ATPase activity. Loss of Ter94 enables the suppression of Hippo target genes. When Ter94 is depleted, it results in reduced wing size and increased apoptosis, which can be rescued by inhibiting the Hippo pathway. Biochemical experiments reveal that Ter94 reciprocally binds to Mer, a critical upstream component of the Hippo pathway, and disrupts its interaction with Ex and Kib. This disruption prevents the formation of the Ex-Mer-Kib complex, ultimately leading to the inactivation of the Hippo pathway and promoting proper wing development. Finally, we show that hVCP, the human homolog of Ter94, is able to substitute for Ter94 in modulating Drosophila wing size, underscoring their functional conservation. In conclusion, Ter94 plays a positive role in regulating wing size by interfering with the Ex-Mer-Kib complex, which results in the suppression of the Hippo pathway.

Hybrid speciation driven by multilocus introgression of ecological traits.

Hybridization allows adaptations to be shared among lineages and may trigger the evolution of new species1,2. However, convincing examples of homoploid hybrid speciation remain rare because it is challenging to demonstrate that hybridization was crucial in generating reproductive isolation3. Here we combine population genomic analysis with quantitative trait locus mapping of species-specific traits to examine a case of hybrid speciation in Heliconius butterflies. We show that Heliconius elevatus is a hybrid species that is sympatric with both parents and has persisted as an independently evolving lineage for at least 180,000 years. This is despite pervasive and ongoing gene flow with one parent, Heliconius pardalinus, which homogenizes 99% of their genomes. The remaining 1% introgressed from the other parent, Heliconius melpomene, and is scattered widely across the H. elevatus genome in islands of divergence from H. pardalinus. These islands contain multiple traits that are under disruptive selection, including colour pattern, wing shape, host plant preference, sex pheromones and mate choice. Collectively, these traits place H. elevatus on its own adaptive peak and permit coexistence with both parents. Our results show that speciation was driven by introgression of ecological traits, and that speciation with gene flow is possible with a multilocus genetic architecture.

Regulation of Notch signaling by non-muscle myosin II Zipper in Drosophila.

The Notch pathway is an evolutionarily conserved signaling system that is intricately regulated at multiple levels and it influences different aspects of development. In an effort to identify novel components involved in Notch signaling and its regulation, we carried out protein interaction screens which identified non-muscle myosin II Zipper (Zip) as an interacting partner of Notch. Physical interaction between Notch and Zip was further validated by co-immunoprecipitation studies. Immunocytochemical analyses revealed that Notch and Zip co-localize within same cytoplasmic compartment. Different alleles of zip also showed strong genetic interactions with Notch pathway components. Downregulation of Zip resulted in wing phenotypes that were reminiscent of Notch loss-of-function phenotypes and a perturbed expression of Notch downstream targets, Cut and Deadpan. Further, synergistic interaction between Notch and Zip resulted in highly ectopic expression of these Notch targets. Activated Notch-induced tumorous phenotype of larval tissues was enhanced by over-expression of Zip. Notch-Zip synergy resulted in the activation of JNK pathway that consequently lead to MMP activation and proliferation. Taken together, our results suggest that Zip may play an important role in regulation of Notch signaling.

Dilp8 and its candidate receptor, Drl, are involved in the transdetermination of the Drosophila imaginal disc.

Drosophila imaginal disc cells can change their identity under stress conditions through transdetermination (TD). Research on TD can help elucidate the in vivo process of cell fate conversion. We previously showed that the overexpression of winged eye (wge) induces eye-to-wing TD in the eye disc and that an insulin-like peptide, Dilp8, is then highly expressed in the disc. Although Dilp8 is known to mediate systemic developmental delay via the Lgr3 receptor, its role in TD remains unknown. This study showed that Dilp8 is expressed in specific cells that do not express eye or wing fate markers during Wge-mediated TD and that the loss of Dilp8 impairs the process of eye-to-wing transition. Thus, Dilp8 plays a pivotal role in the cell fate conversion under wge overexpression. Furthermore, we found that instead of Lgr3, another candidate receptor, Drl, is involved in Wge-mediated TD and acts locally in the eye disc cells. We propose a model in which Dilp8-Drl signaling organizes cell fate conversion in the imaginal disc during TD.

Hierarchical morphogenesis of swallowtail butterfly wing scale nanostructures.

The study of color patterns in the animal integument is a fundamental question in biology, with many lepidopteran species being exemplary models in this endeavor due to their relative simplicity and elegance. While significant advances have been made in unraveling the cellular and molecular basis of lepidopteran pigmentary coloration, the morphogenesis of wing scale nanostructures involved in structural color production is not well understood. Contemporary research on this topic largely focuses on a few nymphalid model taxa (e.g., Bicyclus, Heliconius), despite an overwhelming diversity in the hierarchical nanostructural organization of lepidopteran wing scales. Here, we present a time-resolved, comparative developmental study of hierarchical scale nanostructures in Parides eurimedes and five other papilionid species. Our results uphold the putative conserved role of F-actin bundles in acting as spacers between developing ridges, as previously documented in several nymphalid species. Interestingly, while ridges are developing in P. eurimedes, plasma membrane manifests irregular mesh-like crossribs characteristic of Papilionidae, which delineate the accretion of cuticle into rows of planar disks in between ridges. Once the ridges have grown, disintegrating F-actin bundles appear to reorganize into a network that supports the invagination of plasma membrane underlying the disks, subsequently forming an extruded honeycomb lattice. Our results uncover a previously undocumented role for F-actin in the morphogenesis of complex wing scale nanostructures, likely specific to Papilionidae.

Frizzled2 receives WntA signaling during butterfly wing pattern formation.

Butterfly color patterns provide visible and biodiverse phenotypic readouts of the patterning processes. Although the secreted ligand WntA has been shown to instruct the color pattern formation in butterflies, its mode of reception remains elusive. Butterfly genomes encode four homologs of the Frizzled-family of Wnt receptors. Here, we show that CRISPR mosaic knockouts of frizzled2 (fz2) phenocopy the color pattern effects of WntA loss of function in multiple nymphalids. Whereas WntA mosaic clones result in intermediate patterns of reduced size, fz2 clones are cell-autonomous, consistent with a morphogen function. Shifts in expression of WntA and fz2 in WntA crispant pupae show that they are under positive and negative feedback, respectively. Fz1 is required for Wnt-independent planar cell polarity in the wing epithelium. Fz3 and Fz4 show phenotypes consistent with Wnt competitive-antagonist functions in vein formation (Fz3 and Fz4), wing margin specification (Fz3), and color patterning in the Discalis and Marginal Band Systems (Fz4). Overall, these data show that the WntA/Frizzled2 morphogen-receptor pair forms a signaling axis that instructs butterfly color patterning and shed light on the functional diversity of insect Frizzled receptors.

Vermilion and cinnabar are involved in ommochrome pigment biosynthesis in eyes but not wings of Bicyclus anynana butterflies.

If the same pigment is found in different tissues in a body, it is natural to assume that the same metabolic pathways are deployed similarly in each tissue. Here we show that this is not the case for ommochromes, the red and orange pigments found in the eyes and wings of butterflies. We tested the expression and function of vermilion and cinnabar, two known fly genes in the ommochrome pathway, in the development of pigments in the eyes and in the wings of Bicyclus anynana butterflies, both traits having reddish/orange pigments. By using fluorescent in-situ hybridization (HCR3.0) we localized the expression of vermilion and cinnabar in the cytoplasm of pigment cells in the ommatidia but observed no clear expression for either gene on larval and pupal wings. We then disrupted the function of both genes, using CRISPR-Cas9, which resulted in the loss of pigment in the eyes but not in the wings. Using thin-layer chromatography and UV-vis spectroscopy we identified the presence of ommochrome and ommochrome precursors in the orange wing scales and in the hemolymph of pupae. We conclude that the wings either synthesize ommochromes locally, with yet unidentified enzymes or incorporate these pigments synthesized elsewhere from the hemolymph. Different metabolic pathways or transport mechanisms, thus, lead to the presence of ommochromes in the wings and eyes of B. anynana butterflies.

Involvement of the scalloped gene in morphogenesis of the wing margin via regulating cell growth in a hemimetabolous insect Gryllus bimaculatus.

The acquisition of wings was a key event in insect evolution. As hemimetabolous insects were the first group to acquire functional wings, establishing the mechanisms of wing formation in this group could provide useful insights into their evolution. In this study, we aimed to elucidate the expression and function of the gene scalloped (sd), which is involved in wing formation in Drosophila melanogaster, and in Gryllus bimaculatus mainly during postembryonic development. Expression analysis showed that sd is expressed in the tergal edge, legs, antennae, labrum, and cerci during embryogenesis and in the distal margin of the wing pads from at least the sixth instar in the mid to late stages. Because sd knockout caused early lethality, nymphal RNA interference experiments were performed. Malformations were observed in the wings, ovipositor, and antennae. By analyzing the effects on wing morphology, it was revealed that sd is mainly involved in the formation of the margin, possibly through the regulation of cell proliferation. In conclusion, sd might regulate the local growth of wing pads and influence wing margin morphology in Gryllus.

Divergent expression of aristaless1 and aristaless2 during embryonic appendage and pupal wing development in butterflies.

BACKGROUND: Gene duplication events are critical for the evolution of new gene functions. Aristaless is a major regulator of distinct developmental processes. It is most known for its role during appendage development across animals. However, more recently other distinct biological functions have been described for this gene and its duplicates. Butterflies and moths have two copies of aristaless, aristaless1 (al1) and aristaless2 (al2), as a result of a gene duplication event. Previous work in Heliconius has shown that both copies appear to have novel functions related to wing color patterning. Here we expand our knowledge of the expression profiles associated with both ancestral and novel functions of Al1 across embryogenesis and wing pigmentation. Furthermore, we characterize Al2 expression, providing a comparative framework between gene copies within the same species, allowing us to understand the origin of new functions following gene duplication. RESULTS: Our work shows that the expression of both Al1 and Al2 is associated with the ancestral function of sensory appendage (leg, mouth, spines, and eyes) development in embryos. Interestingly, Al1 exhibits higher expression earlier in embryogenesis while the highest levels of Al2 expression are shifted to later stages of embryonic development. Furthermore, Al1 localization appears extranuclear while Al2 co-localizes tightly with nuclei earlier, and then also expands outside the nucleus later in development. Cellular expression of Al1 and Al2 in pupal wings is broadly consistent with patterns observed during embryogenesis. We also describe, for the first time, how Al1 localization appears to correlate with zones of anterior/posterior elongation of the body during embryonic growth, showcasing a possible new function related to Aristaless' previously described role in appendage extension. CONCLUSIONS: Overall, our data suggest that while both gene copies play a role in embryogenesis and wing pigmentation, the duplicates have diverged temporally and mechanistically across those functions. Our study helps clarify principles behind sub-functionalization and gene expression evolution associated with developmental functions following gene duplication events.

aristaless1 has a dual role in appendage formation and wing color specification during butterfly development.

BACKGROUND: Highly diverse butterfly wing patterns have emerged as a powerful system for understanding the genetic basis of phenotypic variation. While the genetic basis of this pattern variation is being clarified, the precise developmental pathways linking genotype to phenotype are not well understood. The gene aristaless, which plays a role in appendage patterning and extension, has been duplicated in Lepidoptera. One copy, aristaless1, has been shown to control a white/yellow color switch in the butterfly Heliconius cydno, suggesting a novel function associated with color patterning and pigmentation. Here we investigate the developmental basis of al1 in embryos, larvae, and pupae using new antibodies, CRISPR/Cas9, RNAi, qPCR assays of downstream targets, and pharmacological manipulation of an upstream activator. RESULTS: We find that Al1 is expressed at the distal tips of developing embryonic appendages consistent with its ancestral role. In developing wings, we observe Al1 accumulation within developing scale cells of white H. cydno during early pupation while yellow scale cells exhibit little Al1 at this time point. Reduced Al1 expression is also associated with yellow scale development in al1 knockouts and knockdowns. We propose that Al1 expression in future white scales might be related to an observed downregulation of the enzyme Cinnabar and other genes that synthesize and transport the yellow pigment, 3-hydroxykynurenine (3-OHK). Finally, we provide evidence that Al1 activation is under the control of Wnt signaling. CONCLUSIONS: We propose a model in which high levels of Al1 during early pupation, which are mediated by Wnt, are important for melanic pigmentation and specifying white portions of the wing while reduced levels of Al1 during early pupation promote upregulation of proteins needed to move and synthesize 3-OHK, promoting yellow pigmentation. In addition, we discuss how the ancestral role of aristaless in appendage extension may be relevant in understanding the cellular mechanism behind color patterning in the context of the heterochrony hypothesis.

Cis-regulatory evolution underlying the changes in wingless expression pattern associated with wing pigmentation of Drosophila.

The co-option of regulatory genes has the potential to play an important role in the evolutionary gain of new traits. However, the changes at the sequence level that underlie such a co-option event are still elusive. We identified the changes in the cis-regulatory sequence of wingless that caused co-option of wingless and led to its expression in new places in Drosophila guttifera, which has unique pigmentation patterns on its wings. The newly gained function of gene expression activation was acquired evolutionarily via a combination of pre-existing sequences containing a putative binding site for SMAD transcription factors that exhibit an ancestral function in driving expression at crossveins, and a sequence that is specific to the lineage leading to D. guttifera.

A multiscale chemical-mechanical model predicts impact of morphogen spreading on tissue growth.

The exact mechanism controlling cell growth remains a grand challenge in developmental biology and regenerative medicine. The Drosophila wing disc tissue serves as an ideal biological model to study mechanisms involved in growth regulation. Most existing computational models for studying tissue growth focus specifically on either chemical signals or mechanical forces. Here we developed a multiscale chemical-mechanical model to investigate the growth regulation mechanism based on the dynamics of a morphogen gradient. By comparing the spatial distribution of dividing cells and the overall tissue shape obtained in model simulations with experimental data of the wing disc, it is shown that the size of the domain of the Dpp morphogen is critical in determining tissue size and shape. A larger tissue size with a faster growth rate and more symmetric shape can be achieved if the Dpp gradient spreads in a larger domain. Together with Dpp absorbance at the peripheral zone, the feedback regulation that downregulates Dpp receptors on the cell membrane allows for further spreading of the morphogen away from its source region, resulting in prolonged tissue growth at a more spatially homogeneous growth rate.

The mitochondrial ribosomal protein mRpL4 regulates Notch signaling.

Mitochondrial ribosomal proteins (MRPs) assemble as specialized ribosome to synthesize mtDNA-encoded proteins, which are essential for mitochondrial bioenergetic and metabolic processes. MRPs are required for fundamental cellular activities during animal development, but their roles beyond mitochondrial protein translation are poorly understood. Here, we report a conserved role of the mitochondrial ribosomal protein L4 (mRpL4) in Notch signaling. Genetic analyses demonstrate that mRpL4 is required in the Notch signal-receiving cells to permit target gene transcription during Drosophila wing development. We find that mRpL4 physically and genetically interacts with the WD40 repeat protein wap and activates the transcription of Notch signaling targets. We show that human mRpL4 is capable of replacing fly mRpL4 during wing development. Furthermore, knockout of mRpL4 in zebrafish leads to downregulated expression of Notch signaling components. Thus, we have discovered a previously unknown function of mRpL4 during animal development.

Heterodimerization-dependent secretion of bone morphogenetic proteins in Drosophila.

Combinatorial signaling is key to instruct context-dependent cell behaviors. During embryonic development, adult homeostasis, and disease, bone morphogenetic proteins (BMPs) act as dimers to instruct specific cellular responses. BMP ligands can form both homodimers or heterodimers; however, obtaining direct evidence of the endogenous localization and function of each form has proven challenging. Here, we make use of precise genome editing and direct protein manipulation via protein binders to dissect the existence and functional relevance of BMP homodimers and heterodimers in the Drosophila wing imaginal disc. This approach identified in situ the existence of Dpp (BMP2/4)/Gbb (BMP5/6/7/8) heterodimers. We found that Gbb is secreted in a Dpp-dependent manner in the wing imaginal disc. Dpp and Gbb form a gradient of heterodimers, whereas neither Dpp nor Gbb homodimers are evident under endogenous physiological conditions. We find that the formation of heterodimers is critical for obtaining optimal signaling and long-range BMP distribution.

The genetic basis of wing spots in Pieris canidia butterflies.

Spots in pierid butterflies and eyespots in nymphalid butterflies are likely non-homologous wing colour pattern elements, yet they share a few features in common. Both develop black scales that depend on the function of the gene spalt, and both might have central signalling cells. This suggests that both pattern elements may be sharing common genetic circuitry. Hundreds of genes have already been associated with the development of nymphalid butterfly eyespot patterns, but the genetic basis of the simpler spot patterns on the wings of pierid butterflies has not been investigated. To facilitate studies of pierid wing patterns, we report a high-quality draft genome assembly for Pieris canidia, the Indian cabbage white. We then conducted transcriptomic analyses of pupal wing tissues sampled from the spot and non-spot regions of P. canidia at 3-6 h post-pupation. A total of 1352 genes were differentially regulated between wing tissues with and without the black spot, including spalt, Krüppel-like factor 10, genes from the Toll, Notch, TGF-ß, and FGFR signalling pathways, and several genes involved in the melanin biosynthetic pathway. We identified 14 genes that are up-regulated in both pierid spots and nymphalid eyespots and propose that spots and eyespots share regulatory modules despite their likely independent origins.

Salivary carbonic anhydrase II in winged aphid morph facilitates plant infection by viruses.

Aphids are the most common insect vector transmitting hundreds of plant viruses. Aphid wing dimorphism (winged vs. wingless) not only showcases the phenotypic plasticity but also impacts virus transmission; however, the superiority of winged aphids in virus transmission over the wingless morph is not well understood. Here, we show that plant viruses were efficiently transmitted and highly infectious when associated with the winged morph of Myzus persicae and that a salivary protein contributed to this difference. The carbonic anhydrase II (CA-II) gene was identified by RNA-seq of salivary glands to have higher expression in the winged morph. Aphids secreted CA-II into the apoplastic region of plant cells, leading to elevated accumulation of H+. Apoplastic acidification further increased the activities of polygalacturonases, the cell wall homogalacturonan (HG)-modifying enzymes, promoting degradation of demethylesterified HGs. In response to apoplastic acidification, plants accelerated vesicle trafficking to enhance pectin transport and strengthen the cell wall, which also facilitated virus translocation from the endomembrane system to the apoplast. Secretion of a higher quantity of salivary CA-II by winged aphids promoted intercellular vesicle transport in the plant. The higher vesicle trafficking induced by winged aphids enhanced dispersal of virus particles from infected cells to neighboring cells, thus resulting in higher virus infection in plants relative to the wingless morph. These findings imply that the difference in the expression of salivary CA-II between winged and wingless morphs is correlated with the vector role of aphids during the posttransmission infection process, which influences the outcome of plant endurance of virus infection.

Chitinase 6 is required for procuticle thickening and organ shape in Drosophila wing.

The polysaccharide chitin is a major scaffolding molecule in the insect cuticle. In order to be functional, both chitin amounts and chitin organization have been shown to be important parameters. Despite great advances in the past decade, the molecular mechanisms of chitin synthesis and organization are not fully understood. Here, we have characterized the function of the Chitinase 6 (Cht6) in the formation of the wing, which is a simple flat cuticle organ, in the fruit fly Drosophila melanogaster. Reduction of Cht6 function by RNA interference during wing development does not affect chitin organization, but entails a thinner cuticle suggesting reduced chitin amounts. This phenotype is opposed to the one reported recently to be caused by reduction of Cht10 expression. Probably as a consequence, cuticle permeability to xenobiotics is enhanced in Cht6-less wings. We also observed massive deformation of these wings. In addition, the shape of the abdomen is markedly changed upon abdominal suppression of Cht6. Finally, we found that suppression of Cht6 transcript levels influences the expression of genes coding for enzymes of the chitin biosynthesis pathway. This finding indicates that wing epidermal cells respond to activity changes of Cht6 probably trying to adjust chitin amounts. Together, in a working model, we propose that Cht6-introduced modifications of chitin are needed for chitin synthesis to proceed correctly. Cuticle thickness, according to our hypothesis, is in turn required for correct organ or body part shape. The molecular mechanisms of this processes shall be characterized in the future.

Ultrabithorax modifies a regulatory network of genes essential for butterfly eyespot development in a wing sector-specific manner.

Nymphalid butterfly species often have a different number of eyespots in forewings and hindwings, but how the hindwing identity gene Ultrabithorax (Ubx) drives this asymmetry is not fully understood. We examined a three-gene regulatory network for eyespot development in the hindwings of Bicyclus anynana butterflies and compared it with the same network previously described for forewings. We also examined how Ubx interacts with each of these three eyespot-essential genes. We found similar genetic interactions between the three genes in fore- and hindwings, but we discovered three regulatory differences: Antennapedia (Antp) merely enhances spalt (sal) expression in the eyespot foci in hindwings, but is not essential for sal activation, as in forewings; Ubx upregulates Antp in all hindwing eyespot foci but represses Antp outside these wing regions; and Ubx regulates sal in a wing sector-specific manner, i.e. it activates sal expression only in the sectors that have hindwing-specific eyespots. We propose a model for how the regulatory connections between these four genes evolved to produce wing- and sector-specific variation in eyespot number.

Hedgehog signaling can enhance glycolytic ATP production in the Drosophila wing disc.

Adenosine triphosphate (ATP) production and utilization is critically important for animal development. How these processes are regulated in space and time during tissue growth remains largely unclear. We used a FRET-based sensor to dynamically monitor ATP levels across a growing tissue, using the Drosophila larval wing disc. Although steady-state levels of ATP are spatially uniform across the wing pouch, inhibiting oxidative phosphorylation reveals spatial differences in metabolic behavior, whereby signaling centers at compartment boundaries produce more ATP from glycolysis than the rest of the tissue. Genetic perturbations indicate that the conserved Hedgehog signaling pathway can enhance ATP production by glycolysis. Collectively, our work suggests the existence of a homeostatic feedback loop between Hh signaling and glycolysis, advancing our understanding of the connection between conserved developmental patterning genes and ATP production during animal tissue development.