Scaling between cell cycle duration and wing growth is regulated by Fat-Dachsous signaling in Drosophila.

The atypical cadherins Fat and Dachsous (Ds) signal through the Hippo pathway to regulate growth of numerous organs, including the Drosophila wing. Here, we find that Ds-Fat signaling tunes a unique feature of cell proliferation found to control the rate of wing growth during the third instar larval phase. The duration of the cell cycle increases in direct proportion to the size of the wing, leading to linear-like growth during the third instar. Ds-Fat signaling enhances the rate at which the cell cycle lengthens with wing size, thus diminishing the rate of wing growth. We show that this results in a complex but stereotyped relative scaling of wing growth with body growth in Drosophila. Finally, we examine the dynamics of Fat and Ds protein distribution in the wing, observing graded distributions that change during growth. However, the significance of these dynamics is unclear since perturbations in expression have negligible impact on wing growth.

Nuclear position and local acetyl-CoA production regulate chromatin state.

Histone acetylation regulates gene expression, cell function and cell fate1. Here we study the pattern of histone acetylation in the epithelial tissue of the Drosophila wing disc. H3K18ac, H4K8ac and total lysine acetylation are increased in the outer rim of the disc. This acetylation pattern is controlled by nuclear position, whereby nuclei continuously move from apical to basal locations within the epithelium and exhibit high levels of H3K18ac when they are in proximity to the tissue surface. These surface nuclei have increased levels of acetyl-CoA synthase, which generates the acetyl-CoA for histone acetylation. The carbon source for histone acetylation in the rim is fatty acid ß-oxidation, which is also increased in the rim. Inhibition of fatty acid ß-oxidation causes H3K18ac levels to decrease in the genomic proximity of genes involved in disc development. In summary, there is a physical mark of the outer rim of the wing and other imaginal epithelia in Drosophila that affects gene expression.

Par3/bazooka binds NICD and promotes notch signaling during Drosophila development.

The conserved bazooka (baz/par3) gene acts as a key regulator of asymmetrical cell divisions across the animal kingdom. Associated Par3/Baz-Par6-aPKC protein complexes are also well known for their role in the establishment of apical/basal cell polarity in epithelial cells. Here we define a novel, positive function of Baz/Par3 in the Notch pathway. Using Drosophila wing and eye development, we demonstrate that Baz is required for Notch signaling activity and optimal transcriptional activation of Notch target genes. Baz appears to act independently of aPKC in these contexts, as knockdown of aPKC does not cause Notch loss-of-function phenotypes. Using transgenic Notch constructs, our data positions Baz activity downstream of activating Notch cleavage steps and upstream of Su(H)/CSL transcription factor complex activity on Notch target genes. We demonstrate a biochemical interaction between NICD and Baz, suggesting that Baz is required for NICD activity before NICD binds to Su(H). Taken together, our data define a novel role of the polarity protein Baz/Par3, as a positive and direct regulator of Notch signaling through its interaction with NICD.

The First Defined Null Allele of the Notch Regulator, a Suppressor of Deltex: Uncovering Its Novel Roles in Drosophila melanogaster Oogenesis.

Suppressor of deltex (Su(dx)) is a Drosophila melanogaster member of the NEDD4 family of the HECT domain E3 ubiquitin ligases. Su(dx) acts as a regulator of Notch endocytic trafficking, promoting Notch lysosomal degradation and the down-regulation of both ligand-dependent and ligand-independent signalling, the latter involving trafficking through the endocytic pathway and activation of the endo/lysosomal membrane. Mutations of Su(dx) result in developmental phenotypes in the Drosophila wing that reflect increased Notch signalling, leading to gaps in the specification of the wing veins, and Su(dx) functions to provide the developmental robustness of Notch activity to environmental temperature shifts. The full developmental functions of Su(dx) are unclear; however, this is due to a lack of a clearly defined null allele. Here we report the first defined null mutation of Su(dx), generated by P-element excision, which removes the complete open reading frame. We show that the mutation is recessive-viable, with the Notch gain of function phenotypes affecting wing vein and leg development. We further uncover new roles for Su(dx) in Drosophila oogenesis, where it regulates interfollicular stalk formation, egg chamber separation and germline cyst enwrapment by the follicle stem cells. Interestingly, while the null allele exhibited a gain in Notch activity during oogenesis, the previously described Su(dx)SP allele, which carries a seven amino acid in-frame deletion, displayed a Notch loss of function phenotypes and an increase in follicle stem cell turnover. This is despite both alleles displaying similar Notch gain of function in wing development. We attribute this unexpected context-dependent outcome of Su(dx)sp being due to the partial retention of function by the intact C2 and WW domain regions of the protein. Our results extend our understanding of the developmental role of Su(dx) in the tissue renewal and homeostasis of the Drosophila ovary and illustrate the importance of examining an allelic series of mutations to fully understand developmental functions.

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.

Organ transformation by environmental disruption of protein integrity and epigenetic memory in Drosophila.

Despite significant progress in understanding epigenetic reprogramming of cells, the mechanistic basis of "organ reprogramming" by (epi-)gene-environment interactions remained largely obscure. Here, we use the ether-induced haltere-to-wing transformations in Drosophila as a model for epigenetic "reprogramming" at the whole organism level. Our findings support a mechanistic chain of events explaining why and how brief embryonic exposure to ether leads to haltere-to-wing transformations manifested at the larval stage and on. We show that ether interferes with protein integrity in the egg, leading to altered deployment of Hsp90 and widespread repression of Trithorax-mediated establishment of active H3K4me3 chromatin marks throughout the genome. Despite this global reduction, Ubx targets and wing development genes preferentially retain higher levels of H3K4me3 that predispose these genes for later up-regulation in the larval haltere disc, hence the wing-like outcome. Consistent with compromised protein integrity during the exposure, the penetrance of bithorax transformations increases by genetic or chemical reduction of Hsp90 function. Moreover, joint reduction in Hsp90 and trx gene dosage can cause bithorax transformations without exposure to ether, supporting an underlying epistasis between Hsp90 and trx loss-of-functions. These findings implicate environmental disruption of protein integrity at the onset of histone methylation with altered epigenetic regulation of developmental patterning genes. The emerging picture provides a unique example wherein the alleviation of the Hsp90 "capacitor function" by the environment drives a morphogenetic shift towards an ancestral-like body plan. The morphogenetic impact of chaperone response during a major setup of epigenetic patterns may be a general scheme for organ transformation by environmental cues.

Novel Genome-Engineered H Alleles Differentially Affect Lateral Inhibition and Cell Dichotomy Processes during Bristle Organ Development.

Hairless (H) encodes the major antagonist in the Notch signaling pathway, which governs cellular differentiation of various tissues in Drosophila. By binding to the Notch signal transducer Suppressor of Hairless (Su(H)), H assembles repressor complexes onto Notch target genes. Using genome engineering, three new H alleles, HFA, HLLAA and HWA were generated and a phenotypic series was established by several parameters, reflecting the residual H-Su(H) binding capacity. Occasionally, homozygous HWA flies develop to adulthood. They were compared with the likewise semi-viable HNN allele affecting H-Su(H) nuclear entry. The H homozygotes were short-lived, sterile and flightless, yet showed largely normal expression of several mitochondrial genes. Typical for H mutants, both HWA and HNN homozygous alleles displayed strong defects in wing venation and mechano-sensory bristle development. Strikingly, however, HWA displayed only a loss of bristles, whereas bristle organs of HNN flies showed a complete shaft-to-socket transformation. Apparently, the impact of HWA is restricted to lateral inhibition, whereas that of HNN also affects the respective cell type specification. Notably, reduction in Su(H) gene dosage only suppressed the HNN bristle phenotype, but amplified that of HWA. We interpret these differences as to the role of H regarding Su(H) stability and availability.

Local Ecdysone synthesis in a wounded epithelium sustains developmental delay and promotes regeneration in Drosophila.

Regenerative ability often declines as animals mature past embryonic and juvenile stages, suggesting that regeneration requires redirection of growth pathways that promote developmental growth. Intriguingly, the Drosophila larval epithelia require the hormone ecdysone (Ec) for growth but require a drop in circulating Ec levels to regenerate. Examining Ec dynamics more closely, we find that transcriptional activity of the Ec-receptor (EcR) drops in uninjured regions of wing discs, but simultaneously rises in cells around the injury-induced blastema. In parallel, blastema depletion of genes encoding Ec biosynthesis enzymes blocks EcR activity and impairs regeneration but has no effect on uninjured wings. We find that local Ec/EcR signaling is required for injury-induced pupariation delay following injury and that key regeneration regulators upd3 and Ets21c respond to Ec levels. Collectively, these data indicate that injury induces a local source of Ec within the wing blastema that sustains a transcriptional signature necessary for developmental delay and tissue repair.

Temporal dynamics of apoptosis-induced proliferation in pupal wing development: implications for regenerative ability.

BACKGROUND: The ability of animals to regenerate damaged tissue is a complex process that involves various cellular mechanisms. As animals age, they lose their regenerative abilities, making it essential to understand the underlying mechanisms that limit regenerative ability during aging. Drosophila melanogaster wing imaginal discs are epithelial structures that can regenerate after tissue injury. While significant research has focused on investigating regenerative responses during larval stages our comprehension of the regenerative potential of pupal wings and the underlying mechanisms contributing to the decline of regenerative responses remains limited. RESULTS: Here, we explore the temporal dynamics during pupal development of the proliferative response triggered by the induction of cell death, a typical regenerative response. Our results indicate that the apoptosis-induced proliferative response can continue until 34 h after puparium formation (APF), beyond this point cell death alone is not sufficient to induce a regenerative response. Under normal circumstances, cell proliferation ceases around 24 h APF. Interestingly, the failure of reinitiating the cell cycle beyond this time point is not attributed to an incapacity to activate the JNK pathway. Instead, our results suggest that the function of the ecdysone-responsive transcription factor E93 is involved in limiting the apoptosis-induced proliferative response during pupal development. CONCLUSIONS: Our study shows that apoptosis can prolong the proliferative period of cells in the wing during pupal development as late as 34 h APF, at least 10 h longer than during normal development. After this time point, the regenerative response is diminished, a process mediated in part by the ecdysone-responsive transcription factor E93.

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.

Research Note: SOCS2 contributes to reduction of the third digit during development of the chicken forelimb.

The development of the avian wing pattern has been the subject of heated debate due to its special shape. The Suppressor of cytokine signaling 2 (SOCS2) gene encodes a negative regulator of growth hormone (GH) signaling and bone growth and is known to be strongly expressed in the third digit of chicken forelimbs. These observations suggest that SOCS2 might regulate the morphology of the avian wing, however, the function of SOCS2 in avian limb development remains unknown. Here, we reexamined SOCS2 expression in successive developmental stages of chicken limb development by in situ hybridization (ISH) and describe extended expression from the posterior of the stypolod to the third digit of the forelimbs. We used the RCAS avian retrovirus to overexpress SOCS2 in the developing chicken limb buds, which resulted in reduced or malformed chicken wings while hindlimbs developed normally. Transcriptome sequencing (mRNA-Seq) revealed changes in expression of genes known to be associated with growth and development in forelimbs with overexpressed SOCS2. This study highlights a pivotal role for SOCS2 during the development of the wing in the chicken and provides new insight into molecular mechanisms regulating avian limb development.

Role of CYP311A1 in wing development of Drosophila melanogaster.

Lipid homeostasis is crucial for growth and development of organisms. Several cytochrome P450 monooxygenases (CYPs) are involved in lipid metabolism. The function of Cyp311a1 in the anterior midgut as a regulator of phosphatidylethanolamine (PE) metabolism in Drosophila melanogaster has been demonstrated, as depletion of Cyp311a1 caused larval growth arrest that was partially rescued by supplying PE. In this study, we investigated the role of CYP311A1 in wing morphogenesis in Drosophila. Using the GAL4-UAS system, Cyp311a1 was selectively knocked down in the wing disc. A deformed wing phenotype was observed in flies with reduced Cyp311a1 transcripts. BODIPY and oil red O staining revealed a reduction of neutral lipids in the wing disc after the depletion of Cyp311a1. In addition, we observed an enhanced sensitivity to Eosin Y penetration in the wings of Cyp311a1 knocked-down flies. Moreover, the reduction of CYP311A1 function in developing wings does not affect cell proliferation and apoptosis, but entails disordered Phalloidin or Cadherin distribution, suggesting an abnormal cell morphology and cell cortex structure in wing epithelial cells. Taken together, our results suggest that Cyp311a1 is needed for wing morphogenesis by participating in lipid assembly and cell homeostasis.

Elevated developmental temperatures impact the size and allometry of morphological traits of the bumblebee Bombus terrestris.

The impact of global warming on wild bee decline threatens the pollination services they provide. Exposure to temperatures above optimal during development is known to reduce adult body size but how it affects the development and scaling of body parts remains unclear. In bees, a reduction in body size and/or a reduction in body parts, such as the antennae, tongue and wings, and how they scale with body size (i.e. their allometry) could severely affect their fitness. To date, it remains unclear how temperature affects body size and the scaling of morphological traits in bees. To address this knowledge gap, we exposed both males and workers of Bombus terrestris to elevated temperature during development and assessed the effects on (i) the size of morphological traits and (ii) the allometry between these traits. Colonies were exposed to optimal (25°C) or stressful (33°C) temperatures. We then measured the body size, wing size, antenna and tongue length, as well as the allometry between these traits. We found that workers were smaller and the antennae of both castes were reduced at the higher temperature. However, tongue length and wing size were not affected by developmental temperature. The allometric scaling of the tongue was also affected by developmental temperature. Smaller body size and antennae could impair both individual and colony fitness, by affecting foraging efficiency and, consequently, colony development. Our results encourage further exploration of how the temperature-induced changes in morphology affect functional traits and pollination efficiency.

High level of novelty under the hood of convergent evolution.

Little is known about the extent to which species use homologous regulatory architectures to achieve phenotypic convergence. By characterizing chromatin accessibility and gene expression in developing wing tissues, we compared the regulatory architecture of convergence between a pair of mimetic butterfly species. Although a handful of color pattern genes are known to be involved in their convergence, our data suggest that different mutational paths underlie the integration of these genes into wing pattern development. This is supported by a large fraction of accessible chromatin being exclusive to each species, including the de novo lineage-specific evolution of a modular optix enhancer. These findings may be explained by a high level of developmental drift and evolutionary contingency that occurs during the independent evolution of mimicry.

Butterfly eyespots exhibit unique patterns of open chromatin.

Background: How the precise spatial regulation of genes is correlated with spatial variation in chromatin accessibilities is not yet clear. Previous studies that analysed chromatin from homogenates of whole-body parts of insects found little variation in chromatin accessibility across those parts, but single-cell studies of Drosophila brains showed extensive spatial variation in chromatin accessibility across that organ. In this work we studied the chromatin accessibility of butterfly wing tissue fated to differentiate distinct colors and patterns in pupal wings of Bicyclus anynana. Methods: We dissected small eyespot and adjacent control tissues from 3h pupae and performed ATAC-Seq to identify the chromatin accessibility differences between different sections of the wings. Results: We observed that three dissected wing regions showed unique chromatin accessibilities. Open chromatin regions specific to eyespot color patterns were highly enriched for binding motifs recognized by Suppressor of Hairless (Su(H)), Krüppel (Kr), Buttonhead (Btd) and Nubbin (Nub) transcription factors. Genes in the vicinity of the eyespot-specific open chromatin regions included those involved in wound healing and SMAD signal transduction pathways, previously proposed to be involved in eyespot development. Conclusions: We conclude that eyespot and non-eyespot tissue samples taken from the same wing have distinct patterns of chromatin accessibility, possibly driven by the eyespot-restricted expression of potential pioneer factors, such as Kr.

Deep cis-regulatory homology of the butterfly wing pattern ground plan.

Butterfly wing patterns derive from a deeply conserved developmental ground plan yet are diverse and evolve rapidly. It is poorly understood how gene regulatory architectures can accommodate both deep homology and adaptive change. To address this, we characterized the cis-regulatory evolution of the color pattern gene WntA in nymphalid butterflies. Comparative assay for transposase-accessible chromatin using sequencing (ATAC-seq) and in vivo deletions spanning 46 cis-regulatory elements across five species revealed deep homology of ground plan-determining sequences, except in monarch butterflies. Furthermore, noncoding deletions displayed both positive and negative regulatory effects that were often broad in nature. Our results provide little support for models predicting rapid enhancer turnover and suggest that deeply ancestral, multifunctional noncoding elements can underlie rapidly evolving trait systems.

How butterfly wings got their pattern.

Gene regulatory elements play a crucial role in the pattern formation of butterfly wings.

Analysis of the Drosophila Ajuba LIM protein defines functions for distinct LIM domains.

The Ajuba LIM protein Jub mediates regulation of Hippo signaling by cytoskeletal tension through interaction with the kinase Warts and participates in feedback regulation of junctional tension through regulation of the cytohesin Steppke. To investigate how Jub interacts with and regulates its distinct partners, we investigated the ability of Jub proteins missing different combinations of its three LIM domains to rescue jub phenotypes and to interact with α-catenin, Warts and Steppke. Multiple regions of Jub contribute to its ability to bind α-catenin and to localize to adherens junctions in Drosophila wing imaginal discs. Co-immunoprecipitation experiments in cultured cells identified a specific requirement for LIM2 for binding to Warts. However, in vivo, both LIM1 and LIM2, but not LIM3, were required for regulation of wing growth, Yorkie activity, and Warts localization. Conversely, LIM2 and LIM3, but not LIM1, were required for regulation of cell shape and Steppke localization in vivo, and for maximal Steppke binding in co-immunoprecipitation experiments. These observations identify distinct functions for the different LIM domains of Jub.

Roles of Drosophila fatty acid-binding protein in development and behavior.

Fatty acid-binding proteins (FABPs) are lipid chaperones that mediate the intracellular dynamics of the hydrophobic molecules that they physically bind to. FABPs are implicated in sleep and psychiatric disorders, as well as in various cellular processes, such as cell proliferation and survival. FABP is well conserved in insects, and Drosophila has one FABP ortholog, dFabp, in its genome. Although dFabp appears to be evolutionarily conserved in some brain functions, little is known about its development and physiological function. In the present study, we investigated the function of dFabp in Drosophila development and behavior. Knockdown or overexpression of dFabp in the developing brain, wing, and eye resulted in developmental defects, such as decreased survival, altered cell proliferation, and increased apoptosis. Glia-specific knockdown of dFabp affected neuronal development, and neuronal regulation of dFabp affected glial cell proliferation. Moreover, the behavioral phenotypes (circadian rhythm and locomotor activity) of flies with regulated dFabp expression in glia and flies with regulated dFabp expression in neurons were very similar. Collectively, our results suggest that dFabp is involved in the development of various tissues and brain functions to control behavior and is a mediator of neuron-glia interactions in the Drosophila nervous system.

Role of Groucho and Groucho1-like in Regulating Metamorphosis and Ovary Development in Nilaparvata lugens (Stål).

Juvenile hormone and ecdysone are key regulators in the metamorphosis and development. Grocho (Gro) is a highly conserved protein required for metamorphosis and development. Brown planthopper (Nilaparvata lugens) is a major pest affecting rice production in China and many Asian countries. Although the molecular function of Gro has been investigated in holometabolous insects such as Aedes aegypti and Drosophila melanogaster, their role in the hemimetabolous insect, brown planthopper, and the relationship between NlGro/NlGro1-L and JH/ecdysone signaling pathway, remained unknown. In this study, NlGroucho (NlGro) and NlGroucho1-like (NlGro1-L) were cloned. An analysis of the predicted protein sequence showed that NlGro has highly conserved Q domain and WD40 domain, and NlGro1-L has a highly conserved WD40 domain. The expression profiles of both genes were studied by quantitative real-time PCR (qRT-PCR). Their relative expressions were high in egg, head, wing, ovary, and testis. NlGro and NlGro1-L were found to interact genetically with juvenile hormone and ecdysone signaling by hormone treatment and RNAi of JH/ecdysone signaling-related genes. Moreover, when NlGro or NlGro1-L was down-regulated alone, the survival rate was decreased, the ovarian development was delayed, and the oviposition was also affected. All defects were aggravated when NlGro and NlGro1-L were down-regulated together. This study will help to develop new pesticides on the basis of the function of NlGro and NlGro1-L, and provide new possibilities for the control of Nilaparvata lugens.