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Dpp/BMP acts as a morphogen to provide positional information in the Drosophila wing disc. Key cell-surface molecules to control Dpp morphogen gradient formation and signaling are heparan sulfate proteoglycans (HSPGs). In the wing disc, two HSPGs, the glypicans Division abnormally delayed (Dally) and Dally-like (Dlp) have been suggested to act redundantly to control these processes through direct interaction of their heparan sulfate (HS) chains with Dpp. Based on this assumption, a number of models on how glypicans control Dpp gradient formation and signaling have been proposed, including facilitating or hindering Dpp spreading, stabilizing Dpp on the cell surface, or recycling Dpp. However, how distinct HSPGs act remains largely unknown. Here, we generate genome-engineering platforms for the two glypicans and find that only Dally is critical for Dpp gradient formation and signaling through interaction of its core protein with Dpp. We also find that this interaction is not sufficient and that the HS chains of Dally are essential for these functions largely without interacting with Dpp. We provide evidence that the HS chains of Dally are not essential for spreading or recycling of Dpp but for stabilizing Dpp on the cell surface by antagonizing receptor-mediated Dpp internalization. These results provide new insights into how distinct HSPGs control morphogen gradient formation and signaling during development.

Gradients of decapentaplegic (Dpp) pattern Drosophila wing imaginal discs, establishing gene expression boundaries at specific locations. As discs grow, Dpp gradients expand, keeping relative boundary positions approximately stationary. Such scaling fails in mutants for Pentagone (pent), a gene repressed by Dpp that encodes a diffusible protein that expands Dpp gradients. Although these properties fit a recent mathematical model of automatic gradient scaling, that model requires an expander that spreads with minimal loss throughout a morphogen field. Here, we show that Pent's actions are confined to within just a few cell diameters of its site of synthesis and can be phenocopied by manipulating non-diffusible Pent targets strictly within the Pent expression domain. Using genetics and mathematical modeling, we develop an alternative model of scaling driven by feedback downregulation of Dpp receptors and co-receptors. Among the model's predictions is a size beyond which scaling fails-something we observe directly in wing discs.

Wing dimorphism in insects is an evolutionarily adaptive trait to maximize insect fitness under various environments, by which the population could be balanced between dispersing and reproduction. Most studies concern the regulatory mechanisms underlying the stimulation of wing morph in aphids, but relatively little research addresses the molecular basis of wing loss. Here, we found that, while developing normally in winged-destined pea aphids, the wing disc in wingless-destined aphids degenerated 30-hr postbirth and that this degeneration was due to autophagy rather than apoptosis. Activation of autophagy in first instar nymphs reduced the proportion of winged aphids, and suppression of autophagy increased the proportion. REPTOR2, associated with TOR signaling pathway, was identified by RNA-seq as a differentially expressed gene between the two morphs with higher expression in the thorax of wingless-destined aphids. Further genetic analysis indicated that REPTOR2 could be a novel gene derived from a gene duplication event that occurred exclusively in pea aphids on autosome A1 but translocated to the sex chromosome. Knockdown of REPTOR2 reduced autophagy in the wing disc and increased the proportion of winged aphids. In agreement with REPTOR’s canonical negative regulatory role of TOR on autophagy, winged-destined aphids had higher TOR expression in the wing disc. Suppression of TOR activated autophagy of the wing disc and decreased the proportion of winged aphids, and vice versa. Co-suppression of TOR and REPTOR2 showed that dsREPTOR2 could mask the positive effect of dsTOR on autophagy, suggesting that REPTOR2 acted as a key regulator downstream of TOR in the signaling pathway. These results revealed that the TOR signaling pathway suppressed autophagic degradation of the wing disc in pea aphids by negatively regulating the expression of REPTOR2.

Morphogens determine cellular differentiation in many developing tissues in a concentration dependent manner. As a central model for gradient formation during animal development, Hedgehog (Hh) morphogens spread away from their source to direct growth and pattern formation in the Drosophila wing disc. Although heparan sulfate (HS) expression in the disc is essential for this process, it is not known whether HS regulates Hh signaling and spread in a direct or in an indirect manner. To answer this question, we systematically screened two composite Hh binding areas for HS in vitro and expressed mutated proteins in the Drosophila wing disc. We found that selectively impaired HS binding of the second site reduced Hh signaling close to the source and caused striking wing mispatterning phenotypes more distant from the source. These observations suggest that HS constrains Hh to the wing disc epithelium in a direct manner, and that interfering with this constriction converts Hh into freely diffusing forms with altered signaling ranges and impaired gradient robustness.

Animals develop in unpredictable, variable environments. In response to environmental change, some aspects of development adjust to generate plastic phenotypes. Other aspects of development, however, are buffered against environmental change to produce robust phenotypes. How organ development is coordinated to accommodate both plastic and robust developmental responses is poorly understood. Here, we demonstrate that the steroid hormone ecdysone coordinates both plasticity of organ size and robustness of organ pattern in the developing wings of the fruit fly Drosophila melanogaster. Using fed and starved larvae that lack prothoracic glands, which synthesize ecdysone, we show that nutrition regulates growth both via ecdysone and via an ecdysone-independent mechanism, while nutrition regulates patterning only via ecdysone. We then demonstrate that growth shows a graded response to ecdysone concentration, while patterning shows a threshold response. Collectively, these data support a model where nutritionally regulated ecdysone fluctuations confer plasticity by regulating disc growth in response to basal ecdysone levels and confer robustness by initiating patterning only once ecdysone peaks exceed a threshold concentration. This could represent a generalizable mechanism through which hormones coordinate plastic growth with robust patterning in the face of environmental change.

Regeneration is a response mechanism aiming to reconstruct lost or damaged structures. To achieve this, the cells repopulating the lost tissue often have to change their original identity, a process that involves chromatin remodelling.We have analysed the issue of chromatin remodelling during regeneration in the wing disc of Drosophila. In this disc the ablation of the central region (the pouch) induces the regenerative response of the cells from the lateral region (the hinge), which reconstitute the wing pouch. We have examined euchromatin and heterochromatin histone marks during the process and find that heterochromatin marks disappear but are recovered when regeneration is complete. Euchromatin marks are not modified. We also describe the transcription of two retrotransposons, Roo and F-element in the regenerating cells. We have established a temporal correlation between the alterations of heterochromatin marks and the levels of transcription of two retrotransposons, Roo and F-element, both during embryonic development and in the regeneration process. [Display omitted] • During regeneration of the wing disc there is chromatin remodelling, as indicated by histone modifications. • Heterochromatin histone marks are modified during the process but euchromatin marks are not. • The transcription of two retrotransposons, Roo and F-element, is blocked in regenerating cells but resumed at the end of the process. • The epigenetic modifications found during regeneration resemble those that occur during early development. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are post-transcriptional regulators that target specific mRNAs for repression and thus play key roles in many biological processes, including insect wing morphogenesis. miR-2 is an invertebrate-specific miRNA family that has been predicted in the fruit fly, Drosophila melanogaster, to be involved in regulating the Notch signaling pathway. We show here that miR-2 plays a critical role in wing morphogenesis in the silkworm, Bombyx mori, a lepidopteran model insect. Transgenic over-expression of a miR-2 cluster using a Gal4/UAS system results in deformed adult wings, supporting the conclusion that miR-2 regulates functions essential for normal wing morphogenesis. Two genes, abnormal wing disc (awd) and fringe (fng), which are positive regulators in Notch signaling, are identified as miR-2 targets and validated by a dual-luciferase reporter assay. The relative abundance of both awd and fng expression products was reduced significantly in transgenic animals, implicating them in the abnormal wing phenotype. Furthermore, somatic mutagenesis analysis of awd and fng using the CRISPR/Cas9 system and knock-out mutants also resulted in deformed wings similar to those observed in the miR-2 overexpression transgenic animals. The critical role of miR-2 in Bombyx wing morphogenesis may provide a potential target in future lepidopteran pest control.

The growth of imaginal discs in holometabolous insects is coordinated with larval growth to ensure the symmetrical and proportional development of the adult appendages. In ants, the differential growth of these discs generates distinct castes‐the winged male and queen castes and the wingless worker caste. In the hyperdiverse ant genus Pheidole, the worker caste is composed of two morphologically distinct subcastes: small‐headed minor workers and larger, big‐headed, soldiers. Although these worker subcastes are completely wingless, soldier larvae develop rudimentary forewing discs that function in generating the disproportionate head‐to‐body scaling and size of soldiers. It remains unclear, however, how rudimentary forewing discs in soldier larvae are coordinated with other imaginal discs. Here we show, using quantitative nano‐CT imaging and three‐dimensional analyses, that the increase in the volume of the soldier rudimentary forewing discs is coordinated with larval size as well as with the increase in the volume of the leg and eye‐antennal (head) discs. However, relative to larval size, we found that when the rudimentary forewing discs appear during the last larval instar, they are relatively smaller but increase in volume faster than that of the head (eye‐antennal) and leg discs. These findings show that the rudimentary wing disc in soldier larvae has evolved novel patterns of inter‐organ coordination as compared with other insects to generate the big‐headed soldier caste in Pheidole. More generally, our study raises the possibility that novel patterns of inter‐organ coordination are a general feature of rudimentary organs that acquire novel regulatory functions during development and evolution. Research highlights: We applied nano‐CT imaging to ant larvae to study the developmental origin and inter‐organ coordination of caste‐specific traits.We show that a rudimentary organ, evolved novel patterns of inter‐organ coordination and regulation to produce the soldier caste in the ant genus Pheidole. [ABSTRACT FROM AUTHOR]

Abstract Background We classified cuticular protein genes expressed at prepupal stage in wing discs of Bombyx mori into six groups according to their developmental expression and ecdysone responsiveness. Their expression pattern is suggested to be regulated by ecdysone-responsive transcription factors, whose transcripts showed resemblance with those of cuticular protein gene expression. Result Group1 and Group2 CP genes showed peak expression at stage W2. Group3 CP genes showed high expression at stage W3E and W3L and were upregulated by 20E addition, showing a peak 12 h after 20E pulse treatment. Group4 CP gene transcripts started expression from stage V5 and peaked at stage W3L. Some genes showed significant increase 4 or 6 h after 20E addition and were induced 6 h and showed a peak 18 h after the 20E pulse treatment. Group5 CP gene transcripts peaked at the same stage W3L. Some Group5 genes showed significant increase 6 h after the 20E addition, while others were not induced by the 20E addition. These different sub-groups showed different expression profiles in the feeding stage. Transcripts of this group genes were induced 12 h and showed a peak 18 h after the 20E pulse treatment. Group6 CP genes peaked at the stage P0, were not induced by the 20E addition, and showed a peak 24 h after the 20E pulse treatment. Group3, 4, 5, and 6 CP genes are suggested to be regulated by BHR4, BR-C, E74A, and βFTZ-F1, respectively. ERTFs showed different responsiveness to 20E concentration. BR-C was most and E74A was least insensitive. The addition of cycloheximide inhibited BR-C, E74A, and βFTZ-F1 expression depending on the length of treatment after ecdysone pulse treatment, which suggests that BHR4 induced BR-C, E74A, and βFTZ-F1. Conclusion Expression patterns of CPs were determined by the ecdysone-responsiveness and the related ERTFs expressed in the prepupal stage in B. mori wing discs.

Cell division and tissue growth must be coordinated with development. Defects in these processes are the basis for a number of diseases, including developmental malformations and cancer. We have conducted an unbiased RNAi screen for genes that are required for growth in the Drosophila wing, using GAL4-inducible short hairpin RNA (shRNA) fly strains made by the Drosophila RNAi Screening Center. shRNA expression down the center of the larval wing disc using dpp-GAL4, and the central region of the adult wing was then scored for tissue growth and wing hair morphology. Out of 4,753 shRNA crosses that survived to adulthood, 18 had impaired wing growth. FlyBase and the new Alliance of Genome Resources knowledgebases were used to determine the known or predicted functions of these genes and the association of their human orthologs with disease. The function of eight of the genes identified has not been previously defined in Drosophila. The genes identified included those with known or predicted functions in cell cycle, chromosome segregation, morphogenesis, metabolism, steroid processing, transcription, and translation. All but one of the genes are similar to those in humans, and many are associated with disease. Knockdown of lin-52, a subunit of the Myb-MuvB transcription factor, or βNACtes6, a gene involved in protein folding and trafficking, resulted in a switch from cell proliferation to an endoreplication growth program through which wing tissue grew by an increase in cell size (hypertrophy). It is anticipated that further analysis of the genes that we have identified will reveal new mechanisms that regulate tissue growth during development.

Morphogen signaling contributes to the patterned spatiotemporal expression of genes during development. One mode of regulation of signaling-responsive genes is at the level of transcription. Single-cell quantitative studies of transcription have revealed that transcription occurs intermittently, in bursts. Although the effects of many gene regulatory mechanisms on transcriptional bursting have been studied, it remains unclear how morphogen gradients affect this dynamic property of downstream genes. Here we have adapted single molecule fluorescence in situ hybridization (smFISH) for use in the Drosophila wing imaginal disc in order to measure nascent and mature mRNA of genes downstream of the Wg and Dpp morphogen gradients. We compared our experimental results with predictions from stochastic models of transcription, which indicated that the transcription levels of these genes appear to share a common method of control via burst frequency modulation. Our data help further elucidate the link between developmental gene regulatory mechanisms and transcriptional bursting.

The COP9 signalosome inhibits the activity of Cullin-RING E3 ubiquitin ligases by removing Nedd8 modifications from their Cullin subunits. Neddylation renders these complexes catalytically active, but deneddylation is also necessary for them to exchange adaptor subunits and avoid auto-ubiquitination. Although deneddylation is thought to be the primary function of the COP9 signalosome, additional activities have been ascribed to some of its subunits. We recently showed that COP9 subunits protect the transcriptional repressor and tumor suppressor Capicua from two distinct modes of degradation. Deneddylation by the COP9 signalosome inactivates a Cullin 1 complex that ubiquitinates Capicua following its phosphorylation by MAP kinase in response to Epidermal Growth Factor Receptor signaling. The CSN1b subunit also stabilizes unphosphorylated Capicua to control its basal level, independently of the deneddylase function of the complex. Here we further examine the importance of deneddylation for COP9 functions in vivo. We use an uncleavable form of Nedd8 to show that preventing deneddylation does not reproduce the effects of loss of COP9. In contrast, in the presence of COP9, conjugation to uncleavable Nedd8 renders Cullins unable to promote the degradation of their substrates. Our results suggest that irreversible neddylation prolongs COP9 binding to and inhibition of Cullin-based ubiquitin ligases.

The Drosophila melanogaster peripheral nervous system (PNS) comprises the sensory organs that allow the fly to detect environmental factors such as temperature and pressure. PNS development is a highly specified process where each sensilla originates from a single sensory organ precursor (SOP) cell. One of the major genetic orchestrators of PNS development is Senseless, which encodes a zinc finger transcription factor (Sens). Sens is both necessary and sufficient for SOP differentiation. Senseless expression and SOP number are regulated by the microRNA miR-9a. However, the reciprocal dynamics of Senseless and miR-9a are still obscure. By coupling single-molecule FISH with immunofluorescence, we are able to visualize transcription of the mir-9a locus and expression of Sens simultaneously. During embryogenesis, we show that the expression of mir-9a in SOP cells is rapidly lost as Senseless expression increases. However, this mutually exclusive expression pattern is not observed in the third instar imaginal wing disc, where some Senseless-expressing cells show active sites of mir-9a transcription. These data challenge and extend previous models of Senseless regulation and show complex co-expression dynamics between mir-9a and Senseless. The differences in this dynamic relationship between embryonic and larval PNS development suggest a possible switch in miR-9a function. Our work brings single-cell resolution to the understanding of dynamic regulation of PNS development by Senseless and miR-9a. [ABSTRACT FROM AUTHOR]

Hedgehog (Hh) is an evolutionarily conserved signaling protein that has essential roles in animal development and homeostasis. We investigated Hh signaling in the region of the Drosophila wing imaginal disc that produces Hh and is near the tracheal air sac primordium (ASP) and myoblasts. Hh distributes in concentration gradients in the anterior compartment of the wing disc, ASP and myoblasts, and activates genes in each tissue. Some targets of Hh signal transduction are common to the disc, ASP and myoblasts, whereas others are tissue-specific. Signaling in the three tissues is cytoneme-mediated and cytoneme-dependent. Some ASP cells project cytonemes that receive both Hh and Branchless (Bnl), and some targets regulated by Hh signaling in the ASP are also dependent on Bnl signal transduction. We conclude that the single source of Hh in the wing disc regulates cell type-specific responses in three discreet target tissues. [ABSTRACT FROM AUTHOR]

Insect wings are developed from the wing disc during metamorphosis. Bombyx mori, a model lepidopteran insect, loses flight ability after long‐term domestication from the wild silkworm, Bombyx mandarina. The mw mutant (u11 strain) shows minute wings compared to wild type (e.g., p50 strain) wings. RNA sequencing analysis previously revealed differential Hippo‐pathway‐related gene expression between the u11 and p50 strains. The Hippo pathway is an evolutionarily conserved signaling cascade that controls organ size during development in animals. In this study, the function of BmSd which has been characterized as one of the Hippo‐pathway‐related genes was analyzed for silkworm wing development. We found that mats, warts, and hippo expression levels were higher in u11 compared to p50 wing discs. BmSd (scalloped) expression, which encodes a prominent transcriptional partner to Yorkie (Yki), gradually decreased during the wandering stage in u11, but exhibited the opposite expression pattern in p50. When BmSd was knocked down by small interfering RNA during the wandering stage in the p50 strain, 57.9% of the individuals showed minute wings. Additionally, ex, kibra, and wingless expression levels decreased in the BmSd knockdown mutant. Further, BmSd deletion mediated by clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR‐associated protein 9 induced 50% of individuals with minute wings, a phenotype similar to the mw mutant. This result demonstrates that BmSd plays pivotal roles in silkworm wing development. Our results show that the Hippo signaling pathway participates and plays crucial roles in the regulation of silkworm wing development, and our findings provide a basis for further research on B. mori wing development. [ABSTRACT FROM AUTHOR]

• Identification of DEGs in P7 vs L5, M1 vs P7, M1 vs L5 and WM1 vs M1. • Functional annotation of DEGs with gene ontology (GO). • Stage-specific genes were identified from the L5, P7, M1 and WM1. • Insulin metabolism-related genes with high expression in wild silkworm. The insect wing is developed from the wing imaginal disc which is designed from the embryonic ectoderm. To get insight into gene expression profiles in wing discs of Bombyx mori during metamorphosis, we compared the gene expression in the wing between B. mori and B. mandarina moth through RNA-seq. Out of total valid reads identified from the 5th day of 5th instar larvae of silkworm (L5), 7th day of pupae (P7), 1st day of moth (M1) and 1st day of wild silkworm moth (WM1), 20,092,004, 29,251,647, 24,654,695 and 19,753,089 reads were mapped to the mRNA reference sequences of silkworm, respectively. 9229, 7048, 9268 and 6701 differentially expressed genes (DEGs) were respectively recorded in P7 vs L5, M1 vs P7, M1 vs L5 and WM1 vs M1. Further, the peroxisome, ribosome, endocytosis and oxidative phosphorylation pathways were significantly regulated in the metamorphosis of the silkworm. Our study identified 16 orthologous genes with a positive selection from M1, which might be subjected to artificial selection in the domestication of B. mori and would play vital roles in the flight of B. mandarina. [ABSTRACT FROM AUTHOR]

Ras1 (Ras85D) and Ras2 (Ras64B) are the Drosophila orthologs of human H-Ras/N-Ras/K-Ras and R-Ras1-3 genes, respectively. The function of Ras1 has been thoroughly characterised during Drosophila embryonic and imaginal development, and it is associated with coupling activated trans-membrane receptors with tyrosine kinase activity to their downstream effectors. In this capacity, Ras1 binds and is required for the activation of Raf. Ras1 can also interact with PI3K, and it is needed to achieve maximal levels of PI3K signalling in specific cellular settings. In contrast, the function of the unique Drosophila R-Ras member (Ras2/Ras64B), which is more closely related to vertebrate R-Ras2/TC21, has been only studied through the use of constitutively activated forms of the protein. This pioneering work identified a variety of phenotypes that were related to those displayed by Ras1, suggesting that Ras1 and Ras2 might have overlapping activities. Here we find that Ras2 can interact with PI3K and Raf and activate their downstream effectors Akt and Erk. However, and in contrast to mutants in Ras1 , which are lethal, null alleles of Ras2 are viable in homozygosis and only show a phenotype of reduced wing size and extended life span that might be related to reduced Insulin receptor signalling. • We generated mutations in the Drosophila Ras2 gene, orthologous to human RRas1 , RRas2/TC21 and RRas3. • Loss of Ras2 function does not compromise organism viability, but cause reduced wing size and expanded life span. • Ras2 interact with the InR transducer PI3K. • Activated and overexpressed Ras2 can supply for Ras1 function. • Ras2 participates in one of several complementary mechanisms downstream to InR. [ABSTRACT FROM AUTHOR]

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.

Cell migration is a fundamental cell biological process essential both for normal development and for tissue regeneration after damage. Cells can migrate individually or as a collective. To better understand the genetic requirements for collective migration, we expressed RNA interference (RNAi) against 30 genes in the Drosophila embryonic salivary gland cells that are known to migrate collectively. The genes were selected based on their effect on cell and membrane morphology, cytoskeleton and cell adhesion in cell culture-based screens or in Drosophila tissues other than salivary glands. Of these, eight disrupted salivary gland migration, targeting: Rac2, Rab35 and Rab40 GTPases, MAP kinase-activated kinase-2 (MAPk-AK2), RdgA diacylglycerol kinase, Cdk9, the PDSW subunit of NADH dehydrogenase (ND-PDSW) and actin regulator Enabled (Ena). The same RNAi lines were used to determine their effect during regeneration of X-ray-damaged larval wing discs. Cells translocate during this process, but it remained unknown whether they do so by directed cell divisions, by cell migration or both. We found that RNAi targeting Rac2, MAPk-AK2 and RdgA disrupted cell translocation during wing disc regeneration, but RNAi against Ena and ND-PDSW had little effect. We conclude that, in Drosophila, cell movements in development and regeneration have common as well as distinct genetic requirements.

Manipulating an exogenous or endogenous gene of interest at a defined level is critical for a wide variety of experiments. The Gal4/UAS system has been widely used to direct gene expression for studying complex genetic and biological problems in Drosophila melanogaster and other model organisms. Driven by a given tissue‐specific Gal4, expressing UAS‐transgene or UAS‐RNAi (RNA interference) could be used to up‐ or down‐regulate target gene expression, respectively. However, the efficiency of the Gal4/UAS system is roughly predefined by properties of transposon vector constructs and the insertion site in the transgenic stock. Here, we describe a simple way to modulate optomotor blind (omb) expression levels in its endogenous expression region of the wing disc. We co‐expressed UAS‐omb and UAS‐omb‐RNAi together under the control of dpp‐Gal4 driver which is expressed in the omb expression region of the wing pouch. The repression effect is more sensitive to temperature than that of overexpression. At low temperature, overexpression plays a dominant role but the efficiency is attenuated by UAS‐omb‐RNAi. In contrast, at high temperature RNAi predominates in gene expression regulation. By this strategy, we could manipulate omb expression levels at a moderate level. It allows us to manipulate omb expression levels in the same tissue between overexpression and repression at different stages by temperature control. [ABSTRACT FROM AUTHOR]

Tissue development demands precise control of cell proliferation and organization, which is achieved through multiple conserved signaling pathways and protein complexes in multicellular animals. Epithelia are a ubiquitous tissue type that provide diverse functions including physical protection, barrier formation, chemical exchange, and secretory activity. However, epithelial cells are also a common driver of tumorigenesis; thus, understanding the molecular mechanisms that control their growth dynamics is important in understanding not only developmental mechanisms but also disease. One prominent pathway that regulates epithelial growth is the conserved Hippo/Warts/Yorkie network. Hippo/Warts inactivation, or activating mutations in Yorkie that prevent its phosphorylation (e.g., YkiS168A), drive hyperplastic tissue growth. We recently reported that loss of Mushroom body defect (Mud), a microtubule-associated protein that contributes to mitotic spindle function, restricts YkiS168A-mediated growth in Drosophila imaginal wing disc epithelia. Here we show that Mud loss alters cell cycle progression and triggers apoptosis with accompanying Jun kinase (JNK) activation in YkiS168A-expressing discs. To identify additional molecular insights, we performed RNAseq and differential gene expression profiling. This analysis revealed that Mud knockdown in YkiS168A-expressing discs resulted in a significant downregulation in expression of core basement membrane (BM) and extracellular matrix (ECM) genes, including the type IV collagen gene viking. Furthermore, we found that YkiS168A-expressing discs accumulated increased collagen protein, which was reduced following Mud knockdown. Our results suggest that ECM/BM remodeling can limit untoward growth initiated by an important driver of tumor growth and highlight a potential regulatory link with cytoskeleton-associated genes.

In the present study, we tried to clarify when and how pupal commitment (PT) better to use PC occurs and what is involved in the PT of Bombyx mori. To clarify this, we examined the responsiveness of a wing disc to ecdysone, referring to metamorphosis-related BR-C, development-related Myc and Wnt, and chromatin remodeling-related genes at around the predicted PT stage of the Bombyx wing disc. Wing disc responsiveness to juvenile hormone (JH) and ecdysone was examined using Methoprene and 20-hydroxyecdysone (20E) in vitro. The body weight of B. mori increased after the last larval ecdysis, peaked at Day 5 of the fifth larval instar (D5L5), and then decreased. The responsiveness of the wing disc to JH decreased after the last larval ecdysis up to D3L5. Bmbr-c (the Broad Complex of B. mori) showed enhanced expression in D4L5 wing discs with 20E treatment. Some chromatin remodeler and histone modifier genes (Bmsnr1, Bmutx, and Bmtip60) showed upregulation after being cultured with 20E in D4L5 wing discs. A low concentration of 20E is suggested to induce responsiveness to 20E in D4L5 wing discs. Bmbr-c, Bmsnr1, Bmutx, and Bmtip60 were upregulated after being cultured with a low concentration of 20E in D4L5 wing discs. The expression of Bmmyc and Bmwnt1 did not show a change after being cultured with or without 20E in D4L5 wing discs, while enhanced expression was observed with 20E in D5L5 wing discs. From the present results, we concluded that PT of the wing disc of B. mori occurred beginning on D4L5 with the secretion of low concentrations of ecdysteroids. Bmsnr1, Bmutx, Bmtip60, and BR-C are also involved., (© 2024 Wiley Periodicals LLC.)

The Drosophila body comprises a central part, the trunk, and outgrowths of the trunk, the appendages. Much is known about appendage regeneration, but little about the trunk. As the wing imaginal disc contains a trunk component, the notum, and a wing appendage, we have investigated the response to ablation of these two components. We find that, in contrast with the strong regenerative response of the wing, the notum does not regenerate. Nevertheless, the elimination of the wing primordium elicits a proliferative response of notum cells, but they do not regenerate wing; they form a notum duplicate. Conversely, the wing cells cannot regenerate an ablated notum; they overproliferate and generate a hinge overgrowth. These results suggest that trunk and appendages cannot be reprogrammed to generate each other. Our experiments demonstrate that the proliferative response is mediated by JNK signalling from dying cells, but JNK functions differently in the trunk and the appendages, which may explain their distinct regenerative potential. [ABSTRACT FROM AUTHOR]

The COP9 signalosome removes Nedd8 modifications from the Cullin subunits of ubiquitin ligase complexes, reducing their activity. Here, we show that mutations in the Drosophila COP9 signalosome subunit 1b (CSN1b) gene increase the activity of ubiquitin ligases that contain Cullin 1. Analysis of CSN1b mutant phenotypes revealed a requirement for the COP9 signalosome to prevent ectopic expression of Epidermal growth factor receptor (EGFR) target genes. It does so by protecting Capicua, a transcriptional repressor of EGFR target genes, from EGFR pathway-dependent ubiquitylation by a Cullin 1/SKP1-related A/Archipelago E3 ligase and subsequent proteasomal degradation. The CSN1b subunit also maintains basal Capicua levels by protecting it from a separate mechanism of degradation that is independent of EGFR signaling. As a suppressor of tumor growth and metastasis, Capicua may be an important target of the COP9 signalosome in cancer. [ABSTRACT FROM AUTHOR]

Fluorescence recovery after photobleaching (FRAP) is used to obtain quantitative information about molecular diffusion and binding kinetics at both cell and tissue levels of organization. FRAP models have been proposed to estimate the diffusion coefficients and binding kinetic parameters of species for a variety of biological systems and experimental settings. However, it is not clear what the connection among the diverse parameter estimates from different models of the same system is, whether the assumptions made in the model are appropriate, and what the qualities of the estimates are. Here we propose a new approach to investigate the discrepancies between parameters estimated from different models. We use a theoretical model to simulate the dynamics of a FRAP experiment and generate the data that are used in various recovery models to estimate the corresponding parameters. By postulating a recovery model identical to the theoretical model, we first establish that the appropriate choice of observation time can significantly improve the quality of estimates, especially when the diffusion and binding kinetics are not well balanced, in a sense made precise later. Secondly, we find that changing the balance between diffusion and binding kinetics by changing the size of the bleaching region, which gives rise to different FRAP curves, provides a priori knowledge of diffusion and binding kinetics, which is important for model formulation. We also show that the use of the spatial information in FRAP provides better parameter estimation. By varying the recovery model from a fixed theoretical model, we show that a simplified recovery model can adequately describe the FRAP process in some circumstances and establish the relationship between parameters in the theoretical model and those in the recovery model. We then analyze an example in which the data are generated with a model of intermediate complexity and the parameters are estimated using models of greater or less complexity, and show how sensitivity analysis can be used to improve FRAP model formulation. Lastly, we show how sophisticated global sensitivity analysis can be used to detect over-fitting when using a model that is too complex. [ABSTRACT FROM AUTHOR]

Adults of the wingless bagworm moth, Eumeta variegata, show remarkable sexual dimorphism. Final-instar larvae of the male have invaginated wing discs, whereas those of the female are rudimentary. To determine the best method for culturing the wing discs of E. variegata, which in both sexes are attached to the larval integument, two methods of culturing the larval wing discs are compared. Initially, a stationary culture was used. In these cultures necrotic cells and degeneration of wing discs of males were sporadically observed. By contrast, many small vacuoles were observed in the female wing rudiment under these conditions. In order to overcome some of the problems associated with stationary culture, rotating culture was used and resulted in the wing discs of males and females remaining in good condition. A histological analysis revealed that the wing disc morphology was normal when they were cultured in this way. These results indicate that rotating the culture medium is the better procedure for studying the action of hormones on the differentiation and metamorphosis of reduced wing rudiments in E. variegata.

[Display omitted] • Two rapid cell division stages exist in wing discs of 5th larval instar of B. mori. • Insulin and ecdysone induce the cell division through upregulation of Myc. • PI3K/TOR cascades function throughout the 5th larval instar. • MMP genes upregulated by 20E downregulated by the addition of Myc or Wnt inhibitor. • Cell division at feeding stage is for growth, at spinning stage is for differentiation. Through investigating the two different enhanced cell division stages, we tried to clarify the switch from the growth to differentiation in the wing disc of the last larval instar of Bombyx mori. The response to insulin and 20E in vitro was stage specific. Bmmyc expression in V1 wing discs showed differences after being cultured with and without insulin. Bmmyc expression in V5 wing discs also showed differences after being cultured with and without 20E. Cell cycle–related genes, BmE2F1 and BmcycE , were upregulated with insulin or 20E in cultured wing discs of V1 or V5, respectively. Bmwnt1 and Bmras1 showed upregulation with 20E in cultured wing discs. Bmwnt1 showed upregulation with insulin in cultured wing discs, but Bmras1 did not show clear upregulation with insulin treatment. In contrast, Bmdpp showed upregulation with insulin, but did not show clear upregulation with 20E. The addition of PI3K or TOR inhibitors inhibited the upregulation of Bmmyc expression that was upregulated with insulin or 20E. The upregulation of Bmmyc and Bmwnt1 with insulin or 20E was inhibited with the addition of Myc or Wnt inhibitors, respectively. Genes related to matrix metalloprotease showed upregulation with 20E, and the upregulation was inhibited by the addition of Myc or Wnt inhibitors. From the present results, we concluded that cell division during the feeding stage occurred through PI3K/TOR cascade, and that at the wandering stage occurred through ecdysone and PI3K/TOR cascade; the former is for growth and the latter for differentiation. [ABSTRACT FROM AUTHOR]

The insect exoskeleton is mainly composed of chitin filaments linked by cuticle proteins. When insects molt, the cuticle of the exoskeleton is renewed by degrading the old chitin and cuticle proteins and synthesizing new ones. In this study, chitin-binding activity of the wing disc cuticle protein BmWCP4 in Bombyx mori was studied. Sequence analysis showed that the protein had a conservative hydrophilic 'R&R' chitin-binding domain (CBD). Western blotting showed that BmWCP4 was predominately expressed in the wing disc-containing epidermis during the late wandering and early pupal stages. The immunohistochemistry result showed that the BmWCP4 was mainly present in the wing disc tissues containing wing bud and trachea blast during day 2 of wandering stage. Recombinant full-length BmWCP4 protein, 'R&R' CBD peptide (CBD), non-CBD peptide (BmWCP4-CBD−), four single site-directed mutated peptides (M1, M2, M3 and M4) and four-sites-mutated peptide (MF) were generated and purified, respectively, for in vitro chitin-binding assay. The results indicated that both the full-length protein and the 'R&R' CBD peptide could bind with chitin, whereas the BmWCP4-CBD− could not bind with chitin. The single residue mutants M1, M2, M3 and M4 reduced but did not completely abolish the chitin-binding activity, while four-sites-mutated protein MF completely lost the chitin-binding activity. These data indicate that BmWCP4 protein plays a critical role by binding to the chitin filaments in the wing during larva-to-pupa transformation. The conserved aromatic amino acids are critical in the interaction between chitin and the cuticle protein. [ABSTRACT FROM AUTHOR]

Animal morphogenesis requires folds or clefts to separate populations of cells which are often associated with different cell affinities. In the Drosophila wing imaginal disc, the regional expression of the Iroquois complex ( Iro-C ) in the notum leads to the formation of the hinge/notum (H/N) fold that separates the wing hinge and notum territories. Although Decapentaplegic (Dpp) signaling has been revealed as essential for the hinge/notum subdivision through the restriction of Iro-C toward the notum region, the mechanism by which the H/N border develops into a fold is unknown. Here, we report that a Dpp target gene, optomotor-blind ( omb ), mediates the role of Dpp signaling in Iro-C inhibition. omb is complementarily expressed on the dorsal hinge side, abutting the Iro-C domain along the H/N border. Ectopic omb expression inhibits Iro-C in the notum territory, independent of known Iro-C regulators Msh and Stat92E. Uniform manipulation of either omb or Iro-C genes spanning the presumptive H/N border significantly suppresses H/N fold formation via inhibition of the apical microtubule enrichment. Ectopically sharp border or discontinuity in level of Iro-C or Omb is enough to generate ectopic fold formation. These results reveal that omb and Iro-C not only are complementarily expressed but also cooperate to promote H/N fold formation. Our data help to understand how Dpp signaling is interpreted region-specifically during tissue subdivision. [ABSTRACT FROM AUTHOR]

Developmental switching from growth to metamorphosis in imaginal primordia is an essential process of adult body planning in holometabolous insects. Although it is disciplined by a sequential action of the ecdysteroid, molecular mechanisms linking to cell proliferation are poorly understood. In the present study, we investigated the expression control of cell cycle–related genes by the ecdysteroid using the wing disc of the final-instar larvae of the silkworm, Bombyx mori . We found that the expression level of c - myc was remarkably elevated in the post-feeding cell proliferation phase, which coincided with a small increase in ecdysteroid titer. An in vitro wing disc culture showed that supplementation of the moderate level of the ecdysteroid upregulated c - myc expression within an hour and subsequently increased the expression of cell cycle core regulators, including A-, B-, D-, and E-type cyclin genes, Cdc25 and E2F1 . We demonstrated that c - myc upregulation by the ecdysteroid was not inhibited in the presence of a protein synthesis inhibitor, suggesting a possibility that the ecdysteroid directly stimulates c - myc expression. Finally, results from the administration of a c-Myc inhibitor demonstrated that c-Myc plays an essential role in 20E-inducible cell proliferation. These findings suggested a novel pathway for ecdysteroid-inducible cell proliferation in insects, and it is likely to be conserved between insects and mammals in terms of steroid hormone regulation. [ABSTRACT FROM AUTHOR]