A Tumor-Specific Molecular Network Promotes Tumor Growth in Drosophila by Enforcing a Jun N-Terminal Kinase-Yorkie Feedforward Loop.

Cancer cells expand rapidly in response to altered intercellular and signaling interactions to achieve the hallmarks of cancer. Impaired cell polarity combined with activated oncogenes is known to promote several hallmarks of cancer, e.g., activating invasion by increased activity of Jun N-terminal kinase (JNK) and sustained proliferative signaling by increased activity of Hippo effector Yorkie (Yki). Thus, JNK, Yki, and their downstream transcription factors have emerged as synergistic drivers of tumor growth through pro-tumor signaling and intercellular interactions like cell competition. However, little is known about the signals that converge onto JNK and Yki in tumor cells and enable tumor cells to achieve the hallmarks of cancer. Here, using mosaic models of cooperative oncogenesis (RasV12,scrib-) in Drosophila, we show that RasV12,scrib- tumor cells grow through the activation of a previously unidentified network comprising Wingless (Wg), Dronc, JNK, and Yki. We show that RasV12,scrib- cells show increased Wg, Dronc, JNK, and Yki signaling, and all these signals are required for the growth of RasV12,scrib- tumors. We report that Wg and Dronc converge onto a JNK-Yki self-reinforcing positive feedback signal-amplification loop that promotes tumor growth. We found that the Wg-Dronc-Yki-JNK molecular network is specifically activated in polarity-impaired tumor cells and not in normal cells, in which apical-basal polarity remains intact. Our findings suggest that the identification of molecular networks may provide significant insights into the key biologically meaningful changes in signaling pathways and paradoxical signals that promote tumorigenesis.

Automated counting of Drosophila imaginal disc cell nuclei.

Automated image quantification workflows have dramatically improved over the past decade, enriching image analysis and enhancing the ability to achieve statistical power. These analyses have proved especially useful for studies in organisms such as Drosophila melanogaster, where it is relatively simple to obtain high sample numbers for downstream analyses. However, the developing wing, an intensively utilized structure in developmental biology, has eluded efficient cell counting workflows due to its highly dense cellular population. Here, we present efficient automated cell counting workflows capable of quantifying cells in the developing wing. Our workflows can count the total number of cells or count cells in clones labeled with a fluorescent nuclear marker in imaginal discs. Moreover, by training a machine-learning algorithm we have developed a workflow capable of segmenting and counting twin-spot labeled nuclei, a challenging problem requiring distinguishing heterozygous and homozygous cells in a background of regionally varying intensity. Our workflows could potentially be applied to any tissue with high cellular density, as they are structure-agnostic, and only require a nuclear label to segment and count cells.

The AMPK-like protein kinases Sik2 and Sik3 interact with Hipk and induce synergistic tumorigenesis in a Drosophila cancer model.

Homeodomain-interacting protein kinases (Hipks) regulate cell proliferation, apoptosis, and tissue development. Overexpression of Hipk in Drosophila causes tumorigenic phenotypes in larval imaginal discs. We find that depletion of Salt-inducible kinases Sik2 or Sik3 can suppress Hipk-induced overgrowth. Furthermore, co-expression of constitutively active forms of Sik2 or Sik3 with Hipk caused significant tissue hyperplasia and tissue distortion, indicating that both Sik2 and Sik3 can synergize with Hipk to promote tumorous phenotypes, accompanied by elevated dMyc, Armadillo/ß-catenin, and the Yorkie target gene expanded. Larvae expressing these hyperplastic growths also display an extended larval phase, characteristic of other Drosophila tumour models. Examination of total protein levels from fly tissues showed that Hipk proteins were reduced when Siks were depleted through RNAi, suggesting that Siks may regulate Hipk protein stability and/or activity. Conversely, expression of constitutively active Siks with Hipk leads to increased Hipk protein levels. Furthermore, Hipk can interact with Sik2 and Sik3 by co-immunoprecipitation. Co-expression of both proteins leads to a mobility shift of Hipk protein, suggesting it is post-translationally modified. In summary, our research demonstrates a novel function of Siks in synergizing with Hipk to promote tumour growth.

The Drosophila ecdysone receptor promotes or suppresses proliferation according to ligand level.

The steroid hormone 20-hydroxy-ecdysone (20E) promotes proliferation in Drosophila wing precursors at low titer but triggers proliferation arrest at high doses. Remarkably, wing precursors proliferate normally in the complete absence of the 20E receptor, suggesting that low-level 20E promotes proliferation by overriding the default anti-proliferative activity of the receptor. By contrast, 20E needs its receptor to arrest proliferation. Dose-response RNA sequencing (RNA-seq) analysis of ex vivo cultured wing precursors identifies genes that are quantitatively activated by 20E across the physiological range, likely comprising positive modulators of proliferation and other genes that are only activated at high doses. We suggest that some of these "high-threshold" genes dominantly suppress the activity of the pro-proliferation genes. We then show mathematically and with synthetic reporters that combinations of basic regulatory elements can recapitulate the behavior of both types of target genes. Thus, a relatively simple genetic circuit can account for the bimodal activity of this hormone.

Recessive embryonic lethal mutations uncovered in heterozygous condition in silkworm semiconsomic strains.

In this study, we found two embryonic lethal mutations, t04 lethal (l-t04) and m04 lethal (l-m04), in semiconsomic strains T04 and M04, respectively. In these semiconsomic strains, the entire diploid genome, except for one chromosome 4 of the wild silkworm Bombyx mandarina, is substituted with chromosomes of the domesticated silkworm B. mori, and l-t04 and l-m04 mutations are located on B. mandarina-derived chromosome 4. To clarify the cause of the lethalities and the genes responsible for these mutations, positional cloning and CRISPR/Cas9 mediated knockout screening were performed. Finally, genetic complementation tests identified the mutations responsible for the l-t04 and l-m04 as the Bombyx homolog of imaginal discs arrested (Bmida) and TATA box binding protein-associated factor 5 (BmTaf5), respectively. Lethal stages of each knockout mutant indicated the importance of these genes in B. mori late embryogenesis. The lethal mutations responsible for l-t04 and l-m04 were not found in parental strains or wild B. mandarina collected from 39 distinct locations in Japan, indicating that both mutations were independently introduced during or after the development of the semiconsomic strains. We conclude that the recessive embryonic lethality in the T04 and M04 strains is due to deleterious mutations produced in B. mandarina-derived chromosome 4.

Preparation of a Single-cell Suspension from Drosophila Wing Imaginal Discs.

The wing imaginal discs in Drosophila larvae are a pair of sac-like structures that later form the wings of the adult fly. During the past decades, wing discs have been used as a simple and accessible model system, for identifying genes and deciphering signaling cascades that play crucial roles in many aspects of development. In this protocol, we describe a simple method for preparing a cell suspension from wing discs (see Graphical abstract). This method can also be applied to the preparation of single-cell suspensions from other types of Drosophila tissues. When combined with genetic labeling, the dissociated cells are suitable for downstream analysis, such as flow cytometry. This method was recently used to isolate different populations of cells from Drosophila imaginal discs ( Yang et al., 2022 ). Graphical abstract: Procedures to prepare a single-cell suspension from Drosophila imaginal discs. Illustration of the main steps to dissect, temporarily store, and dissociate imaginal discs from Drosophila larvae. Refer to the Procedure section for detailed description of each step.

Mass Purification Protocol for Drosophila melanogaster Wing Imaginal Discs: An Alternative to Dissection to Obtain Large Numbers of Disc Cells.

Drosophila melanogaster imaginal discs are larval internal structures that become the external organs of the adult. They have been used to study numerous developmental processes for more than fifty years. Dissecting these imaginal discs for collection is challenging, as the size of third-instar larvae organs is typically less than 1 mm. Certain experimental applications of the organs require many cells, which requires researchers to spend several hours dissecting them. This paper proposes an alternative to dissection in the form of a mass enrichment protocol. The protocol enables the recovery of many wing imaginal discs by grinding large quantities of third-instar larvae and separating the organs using filtration and a density gradient. The wing imaginal discs collected with this protocol in less than three hours are as well preserved as those collected by dissection. The dissociation and filtration of the extract allow the isolation of a large amount of wing imaginal disc cells.

Without mechanisms, theories and models in insect epigenetics remain a black box.

Insect epigenetics must confront the remarkable diversity of epigenomic systems in various lineages and use mechanistic approaches to move beyond vague functional explanations based on predictions and inferences. To accelerate progress, what is required now is a convergence of genomic data with biochemical and single-cell-type analyses in selected species representing contrasting evolutionary solutions in epigenetics.

Mechanical constraints to cell-cycle progression in a pseudostratified epithelium.

As organs and tissues approach their normal size during development or regeneration, growth slows down, and cell proliferation progressively comes to a halt. Among the various processes suggested to contribute to growth termination,1-10 mechanical feedback, perhaps via adherens junctions, has been suggested to play a role.11-14 However, since adherens junctions are only present in a narrow plane of the subapical region, other structures are likely needed to sense mechanical stresses along the apical-basal (A-B) axis, especially in a thick pseudostratified epithelium. This could be achieved by nuclei, which have been implicated in mechanotransduction in tissue culture.15 In addition, mechanical constraints imposed by nuclear crowding and spatial confinement could affect interkinetic nuclear migration (IKNM),16 which allows G2 nuclei to reach the apical surface, where they normally undergo mitosis.17-25 To explore how mechanical constraints affect IKNM, we devised an individual-based model that treats nuclei as deformable objects constrained by the cell cortex and the presence of other nuclei. The model predicts changes in the proportion of cell-cycle phases during growth, which we validate with the cell-cycle phase reporter FUCCI (Fluorescent Ubiquitination-based Cell Cycle Indicator).26 However, this model does not preclude indefinite growth, leading us to postulate that nuclei must migrate basally to access a putative basal signal required for S phase entry. With this refinement, our updated model accounts for the observed progressive slowing down of growth and explains how pseudostratified epithelia reach a stereotypical thickness upon completion of growth.

Expression of human HIPKs in Drosophila demonstrates their shared and unique functions in a developmental model.

Homeodomain-interacting protein kinases (HIPKs) are a family of four conserved proteins essential for vertebrate development, as demonstrated by defects in the eye, brain, and skeleton that culminate in embryonic lethality when multiple HIPKs are lost in mice. While HIPKs are essential for development, functional redundancy between the four vertebrate HIPK paralogues has made it difficult to compare their respective functions. Because understanding the unique and shared functions of these essential proteins could directly benefit the fields of biology and medicine, we addressed the gap in knowledge of the four vertebrate HIPK paralogues by studying them in the fruit fly Drosophila melanogaster, where reduced genetic redundancy simplifies our functional assessment. The single hipk present in the fly allowed us to perform rescue experiments with human HIPK genes that provide new insight into their individual functions not easily assessed in vertebrate models. Furthermore, the abundance of genetic tools and established methods for monitoring specific developmental pathways and gross morphological changes in the fly allowed for functional comparisons in endogenous contexts. We first performed rescue experiments to demonstrate the extent to which each of the human HIPKs can functionally replace Drosophila Hipk for survival and morphological development. We then showed the ability of each human HIPK to modulate Armadillo/ß-catenin levels, JAK/STAT activity, proliferation, growth, and death, each of which have previously been described for Hipks, but never all together in comparable tissue contexts. Finally, we characterized novel developmental phenotypes induced by human HIPKs to gain insight to their unique functions. Together, these experiments provide the first direct comparison of all four vertebrate HIPKs to determine their roles in a developmental context.

Transcriptome dynamics during metamorphosis of imaginal discs into wings and thoracic dorsum in Apis mellifera castes.

BACKGROUND: Much of the complex anatomy of a holometabolous insect is built from disc-shaped epithelial structures found inside the larva, i.e., the imaginal discs, which undergo a rapid differentiation during metamorphosis. Imaginal discs-derived structures, like wings, are built through the action of genes under precise regulation. RESULTS: We analyzed 30 honeybee transcriptomes in the search for the gene expression needed for wings and thoracic dorsum construction from the larval wing discs primordia. Analyses were carried out before, during, and after the metamorphic molt and using worker and queen castes. Our RNA-seq libraries revealed 13,202 genes, representing 86.2% of the honeybee annotated genes. Gene Ontology analysis revealed functional terms that were caste-specific or shared by workers and queens. Genes expressed in wing discs and descendant structures showed differential expression profiles dynamics in premetamorphic, metamorphic and postmetamorphic developmental phases, and also between castes. At the metamorphic molt, when ecdysteroids peak, the wing buds of workers showed maximal gene upregulation comparatively to queens, thus underscoring differences in gene expression between castes at the height of the larval-pupal transition. Analysis of small RNA libraries of wing buds allowed us to build miRNA-mRNA interaction networks to predict the regulation of genes expressed during wing discs development. CONCLUSION: Together, these data reveal gene expression dynamics leading to wings and thoracic dorsum formation from the wing discs, besides highlighting caste-specific differences during wing discs metamorphosis.

Mechanics of epidermal morphogenesis in the Drosophila pupa.

Adult epidermal development in Drosophila showcases a striking balance between en masse spreading of the developing adult precursor tissues and retraction of the degenerating larval epidermis. The adult precursor tissues, driven by both intrinsic plasticity and extrinsic mechanical cues, shape the segments of the adult epidermis and appendages. Here, we review the tissue architectural changes that occur during epidermal morphogenesis in the Drosophila pupa, with a particular emphasis on the underlying mechanical principles. We highlight recent developments in our understanding of adult epidermal morphogenesis. We further discuss the forces that drive these morphogenetic events and finally outline open questions and challenges.

A PI4KIIIα protein complex is required for cell viability during Drosophila wing development.

Phosphatidylinositol 4 phosphate (PI4P) and phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] are enriched on the inner leaflet of the plasma membrane and proposed to be key determinants of its function. PI4P is also the biochemical precursor for the synthesis of PI(4,5)P2 but can itself also bind to and regulate protein function. However, the independent function of PI4P at the plasma membrane in supporting cell function in metazoans during development in vivo remains unclear. We find that conserved components of a multi-protein complex composed of phosphatidylinositol 4-kinase IIIα (PI4KIIIα), TTC7 and Efr3 is required for normal vein patterning and wing development. Depletion of each of these three components of the PI4KIIIα complex in developing wing cells results in altered wing morphology. These effects are associated with an increase in apoptosis and can be rescued by expression of an inhibitor of Drosophila caspase. We find that in contrast to previous reports, PI4KIIIα depletion does not alter key outputs of hedgehog signalling in developing wing discs. Depletion of PI4KIIIα results in reduced PI4P levels at the plasma membrane of developing wing disc cells while levels of PI(4,5)P2, the downstream metabolite of PI4P, are not altered. Thus, PI4P itself generated by the activity of the PI4KIIIα complex plays an essential role in supporting cell viability in the developing Drosophila wing disc.

JNK Signaling as a Key Modulator of Soft Connective Tissue Physiology, Pathology, and Healing.

In healthy individuals, the healing of soft tissues such as skin after pathological insult or post injury follows a relatively predictable and defined series of cell and molecular processes to restore tissue architecture and function(s). Healing progresses through the phases of hemostasis, inflammation, proliferation, remodeling, and concomitant with re-epithelialization restores barrier function. Soft tissue healing is achieved through the spatiotemporal interplay of multiple different cell types including neutrophils, monocytes/macrophages, fibroblasts, endothelial cells/pericytes, and keratinocytes. Expressed in most cell types, c-Jun N-terminal kinases (JNK) are signaling molecules associated with the regulation of several cellular processes involved in soft tissue wound healing and in response to cellular stress. A member of the mitogen-activated protein kinase family (MAPK), JNKs have been implicated in the regulation of inflammatory cell phenotype, as well as fibroblast, stem/progenitor cell, and epithelial cell biology. In this review, we discuss our understanding of JNKs in the regulation of cell behaviors related to tissue injury, pathology, and wound healing of soft tissues. Using models as diverse as Drosophila, mice, rats, as well as human tissues, research is now defining important, but sometimes conflicting roles for JNKs in the regulation of multiple molecular processes in multiple different cell types central to wound healing processes. In this review, we focus specifically on the role of JNKs in the regulation of cell behavior in the healing of skin, cornea, tendon, gingiva, and dental pulp tissues. We conclude that while parallels can be drawn between some JNK activities and the control of cell behavior in healing, the roles of JNK can also be very specific modes of action depending on the tissue and the phase of healing.

Analysis of the Metaphase Chromosome Karyotypes in Imaginal Discs of Aedes communis, Ae. punctor, Ae. intrudens, and Ae. rossicus (Diptera: Culicidae) Mosquitoes.

In this study, cytogenetic analysis of the metaphase chromosomes from imaginal discs of Aedes (Diptera: Culicidae) mosquitoes-Aedes communis, Ae. punctor, Ae. intrudens, and Ae. rossicus-was performed. The patterns of C-banding and DAPI staining of the heteroсhromatin and the length of the chromosomes demonstrate species specificity. In particular, the Ae. punctor chromosomes are the shortest compared with Ae. communis, Ae. intrudens, and Ae. rossicus, and they also carry additional C and DAPI bands in intercalary regions. The Ae. intrudens chromosomes are the longest, they have pericentromeric C bands, and they almost lack any DAPI bands near the centromere of chromosome 3 versus Ae. communis, which has the largest pericentromeric DAPI blocks in all three chromosome pairs. Ae. rossicus also possesses DAPI bands in the centromeric regions of all chromosomes, but their staining is weaker compared with those of Ae. communis. Therefore, the analysis of karyotypes is a tool for species-level identification of these mosquitoes.

Single cell transcriptomic landscapes of pattern formation, proliferation and growth in Drosophila wing imaginal discs.

Organ formation relies on the orchestration of pattern formation, proliferation and growth during development. How these processes are integrated at the individual cell level remains unclear. In the past decades, studies using Drosophila wing imaginal discs as a model system have provided valuable insights into pattern formation, growth control and regeneration. Here, we provide single cell transcriptomic landscapes of pattern formation, proliferation and growth of wing imaginal discs. We found that patterning information is robustly maintained in the single cell transcriptomic data and can provide reference matrices for computationally mapping single cells into discrete spatial domains. Assignment of wing disc single cells to spatial subregions facilitates examination of patterning refinement processes. We also clustered single cells into different proliferation and growth states and evaluated the correlation between cell proliferation/growth states and spatial patterning. Furthermore, single cell transcriptomic analyses allowed us to quantitatively examine disturbances of differentiation, proliferation and growth in a well-established tumor model. We provide a database to explore these datasets at http://drosophilayanlab-virtual-wingdisc.ust.hk:3838/v2/This article has an associated 'The people behind the papers' interview.

Genetic and Cell Biology Methods to Study ESCRTs in Drosophila melanogaster.

Mosaic analysis in Drosophila represents a convenient entry point for studying the role of ESCRT (Endosomal Sorting Complex Required for Transport) genes in multiple cell processes crucial for organ development and homeostasis. Here, we describe the procedure to generate populations of ESCRT-mutant cells within Drosophila larval epithelial organs and to study them in whole-mount preparations using confocal microscopy. The use of antibodies directed to endocytic cargoes, vesicular trafficking, cell proliferation, death, and polarity markers allows one to investigate the consequences of loss of ESCRT activity at the subcellular and tissue level. The protocols described here can be used in fixed tissue as well as in unfixed tissue using endocytic uptake assays.

Role of D-GADD45 in JNK-Dependent Apoptosis and Regeneration in Drosophila.

The GADD45 proteins are induced in response to stress and have been implicated in the regulation of several cellular functions, including DNA repair, cell cycle control, senescence, and apoptosis. In this study, we investigate the role of D-GADD45 during Drosophila development and regeneration of the wing imaginal discs. We find that higher expression of D-GADD45 results in JNK-dependent apoptosis, while its temporary expression does not have harmful effects. Moreover, D-GADD45 is required for proper regeneration of wing imaginal discs. Our findings demonstrate that a tight regulation of D-GADD45 levels is required for its correct function both, in development and during the stress response after cell death.

Live Imaging of Hippo Pathway Components in Drosophila Imaginal Discs.

Examining the subcellular localization of Hippo pathway components has helped elucidate the molecular mechanisms that regulate the pathway. Here we describe methods for performing live imaging of fluorescently tagged Hippo pathway components in Drosophila wing imaginal discs.

Bimolecular Fluorescence Complementation (BiFC) in Tissue Culture and in Developing Tissues of Drosophila to Study Protein-Protein Interactions.

Protein-protein interactions provide a common mechanism for regulating protein functions and also serve as the fundamental step of many biochemical reactions. To accurately determine the involvement and function of protein-protein interactions, it is crucial to detect the interactions with the minimum number of artifacts. In this chapter, we report the method of bimolecular fluorescence complementation (BiFC) in tissue culture and developing tissues of Drosophila, which allows the visualization of subcellular localization of protein-protein interactions in living cells.