Polyploidy, an increase in cellular genome content, is a conserved developmental program and a hallmark of malignant cancer, yet its impact on cell behavior remains poorly understood. Here, we show that induction of polyploidy in otherwise diploid cells causes intrinsic stress that reprograms cellular physiology to promote motility-like and phagocytic behaviors. Using the Drosophila wing imaginal disc, we find that induced polyploidy enhances membrane dynamics and triggers dynamic cell behavior through a ROS-JNK stress axis activated by ER stress. These cells also acquire phagocytic activity, engulfing both dead and live neighboring cells in developmental and tumor contexts. This stress-induced reprogramming is conserved in induced poly-aneuploid mammalian cancer cells, linking increased genomic content to metastatic traits. Our findings uncover a conserved role for induced polyploidy in driving stress-responsive and immune cell-like behaviors, revealing how elevated ploidy can reshape epithelial function during development and disease.

The integration of metabolic status with reproductive and developmental timing is a cornerstone of animal physiology, yet how steroidogenesis rapidly adapts to abrupt environmental changes remains poorly understood. Here, we identify a peptide hormone circuit in which leptin or its analogs act as peripheral tropic signals, directly coupling systemic metabolic state to steroid hormone production through dynamic remodeling of intracellular lipid pools. Mechanistically, spatiotemporal fluctuations in leptin activate JAK/STAT signaling within steroidogenic tissues to reprogram the balance among lipid droplets, cholesteryl esters, and free cholesterol, thereby tuning the amplitude and timing of steroid hormone pulses via hormone-sensitive lipase (Hsl), a rate-limiting determinant of steroidogenic flux. Cross-species analyses in Drosophila melanogaster, Blattella germanica, and Mus musculus suggest a partially conserved leptin-JAK/STAT-lipase axis that functions as a metabolic "sterol rheostat", enabling rapid modulation of steroidogenesis in response to systemic metabolic and stress cues. These findings reveal a metabolic-endocrine mechanism by which leptin can act directly on steroidogenic organs to regulate hormonal output.

The transformation of epithelial cells into Non-Professional Phagocytes (NPPs) is a conserved and versatile adaptation that occurs in response to immune challenges, tissue remodeling, and apoptotic debris clearance. In the Drosophila melanogaster ovary, follicle cells (FCs) acquire phagocytic capabilities under stress, providing a powerful model to study this process. Using time-lapse live imaging, we captured dynamic behaviors associated with FC-to-NPP differentiation that are too transient to detect by static imaging. Our approach confirmed established features, including germline cell death, cytoplasmic expansion, and debris engulfment, and revealed previously unrecognized capabilities. These include a gradual increase in JNK pathway activation, after which NPPs exhibit collective migration toward dying germline cells, epithelial delamination, long-range target capture through pseudopodial extensions, and the engulfment of neighboring FCs. These findings demonstrate that epithelial-derived NPPs can perform complex phagocytic tasks typically attributed to professional phagocytes such as macrophages. Our work establishes the Drosophila ovary as a robust in vivo system to uncover conserved and novel aspects of epithelial plasticity and phagocytic function, particularly those involving transient behaviors missed by fixed-sample analyses.

The differentiation of epithelial cells into Non-Professional Phagocytes (NPPs) is essential for maintaining tissue homeostasis and clearing apoptotic debris. In the Drosophila ovary, epithelial follicle cells transform into NPPs following germline cell death, but the genetic mechanisms controlling this transition are not well defined. To investigate these mechanisms, we used a model in which overexpression of the active form of Notch, the Notch Intracellular Domain (NICD), induces a robust epithelial-to-NPP transition. Using single-cell RNA sequencing and trajectory analysis, we identified three transcriptional phases of NPP maturation: an early stage of metabolic activation, an intermediate stage enriched in genes related to migration and cytoskeletal remodeling, and a late stage marked by autophagy-related gene expression. These transcriptomic patterns were validated by immunostaining. SCENIC and ChiP-seq analyses identified the JNK effector Jun-related antigen (Jra) and its predicted targets, Arp2 and Arp3, which encode components of the Arp2/3 complex, as regulators of cytoskeletal remodeling. Functional assays confirmed that the JNK-Jra-Arp2/3 axis is required for cytoplasmic expansion and debris clearance during NPP differentiation.

Removal of dead and damaged cells is critical for organismal health. Under stress conditions such as nutritional deprivation, infection, or temperature shift, the clearance of nonessential cells becomes a universal strategy to conserve energy and maintain tissue homeostasis. Typically, this task is performed by professional phagocytes such as macrophages. However, nonprofessional phagocytes (NPPs) can also adopt a phagocytic fate under specific circumstances. Similar to professional phagocytes, NPPs undergo transitions from immature to mature states and activation, but the precise cellular and molecular mechanisms governing their maturation, induction, and phagocytic execution remain largely unknown. A notable example of stress-induced phagocytosis is the removal of germline cells by follicle cell-derived NPPs during oogenesis in Drosophila. In this study, we report that the transformation of follicle cells (FCs) into NPPs is dependent on Notch signaling activation during mid-oogenesis. Moreover, Notch overactivation is sufficient to trigger germline cell death and clearance (GDAC). We further show that polyploidy, driven by Notch signaling-induced endoreplication, is essential for the transformation of FCs into NPPs. Polyploidy facilitates the activation of JNK signaling, which is crucial for the phagocytic behavior of these cells. Additionally, we show that polyploidy in epidermal cells, another type of NPPs, is important for their engulfment of dendrites during induced degeneration. Together, these findings suggest that polyploidy is a critical factor in the transformation of epithelial cells into NPPs, enabling their phagocytic functions, which are essential for maintaining cellular and organismal homeostasis during stress conditions.

Tumor growth and progression involve coordinated regulation by internal, microenvironmental, and systemic signals and often display conspicuous sexual dimorphism. The mechanisms governing the integration and coordination of these signals, along with their sex-based differences, remain largely unknown. Using a Drosophila tumor model originating from nonreproductive tissue, we show that female-biased tumor growth involves multifaceted communications among tumor cells, hemocytes, and neuroendocrine insulin-producing cells (IPCs). Notch-active tumor cells recruit hemocytes carrying the tumor necrosis factor-α (TNF-α) homolog Eiger to the tumor microenvironment (TME), activating the c-Jun N-terminal kinase (JNK) pathway in tumor cells, instigating the sexually dimorphic up-regulation of cytokine Unpaired 2 (Upd2). Upd2, in turn, exerts a distal influence by modulating the release of a Drosophila insulin-like peptide (Dilp2) from IPCs. Dilp2 then activates the insulin signaling in the tumor, thereby fostering sexual-dimorphic tumor growth. Together, these findings reveal a relay mechanism involving the TME and systemic signals that collectively control the sexual dimorphism of tumor growth.

In proliferating neoplasms, microenvironment-derived selective pressures promote tumor heterogeneity by imparting diverse capacities for growth, differentiation, and invasion. However, what makes a tumor cell respond to signaling cues differently from a normal cell is not well understood. In the Drosophila ovarian follicle cells, apicobasal-polarity loss induces heterogeneous epithelial multilayering. When exacerbated by oncogenic-Notch expression, this multilayer displays an increased consistency in the occurrence of morphologically distinguishable cells adjacent to the polar follicle cells. Polar cells release the Jak/STAT ligand Unpaired (Upd), in response to which neighboring polarity-deficient cells exhibit a precursor-like transcriptomic state. Among the several regulons active in these cells, we could detect and further validate the expression of Snail family transcription factor Escargot (Esg). We also ascertain a similar relationship between Upd and Esg in normally developing ovaries, where establishment of polarity determines early follicular differentiation. Overall, our results indicate that epithelial-cell polarity acts as a gatekeeper against microenvironmental selective pressures that drive heterogeneity.

Supplementary Table 1: The mRNA level changes of representative components or targets of JAK-STAT, JNK, and Notch signaling pathways in snr1-RNAi tumorous wing discs relative to control wildtype wing discs.

Legends and methods.

Apical-basal polarity is maintained by distinct protein complexes that reside in membrane junctions, and polarity loss in monolayered epithelial cells can lead to formation of multilayers, cell extrusion, and/or malignant overgrowth. Yet, how polarity loss cooperates with intrinsic signals to control directional invasion toward neighboring epithelial cells remains elusive. Using the Drosophila ovarian follicular epithelium as a model, we found that posterior follicle cells with loss of lethal giant larvae (lgl) or Discs large (Dlg) accumulate apically toward germline cells, whereas cells with loss of Bazooka (Baz) or atypical protein kinase C (aPKC) expand toward the basal side of wildtype neighbors. Further studies revealed that these distinct multilayering patterns in the follicular epithelium were determined by epidermal growth factor receptor (EGFR) signaling and its downstream target Pointed, a zinc-finger transcription factor. Additionally, we identified Rho kinase as a Pointed target that regulates formation of distinct multilayering patterns. These findings provide insight into how cell polarity genes and receptor tyrosine kinase signaling interact to govern epithelial cell organization and directional growth that contribute to epithelial tumor formation.

Sexual attraction and perception, governed by separate genetic circuits in different organs, are crucial for mating and reproductive success, yet the mechanisms of how these two aspects are integrated remain unclear. In Drosophila , the male-specific isoform of Fruitless (Fru), Fru M , is known as a master neuro-regulator of innate courtship behavior to control perception of sex pheromones in sensory neurons. Here we show that the non-sex specific Fru isoform (Fru COM ) is necessary for pheromone biosynthesis in hepatocyte-like oenocytes for sexual attraction. Loss of Fru COM in oenocytes resulted in adults with reduced levels of the cuticular hydrocarbons (CHCs), including sex pheromones, and show altered sexual attraction and reduced cuticular hydrophobicity. We further identify Hepatocyte nuclear factor 4 ( Hnf4 ) as a key target of Fru COM in directing fatty acid conversion to hydrocarbons in adult oenocytes. fru - and Hnf4 -depletion disrupts lipid homeostasis, resulting in a novel sex-dimorphic CHC profile, which differs from doublesex - and transformer -dependent sexual dimorphism of the CHC profile. Thus, Fru couples pheromone perception and production in separate organs for precise coordination of chemosensory communication that ensures efficient mating behavior. Teaser:Fruitless and lipid metabolism regulator HNF4 integrate pheromone biosynthesis and perception to ensure robust courtship behavior.

Supplementary figure 1: Snr1 is required for cell survival in Drosophila wing imaginal disc. Supplementary figure 2: Tumorigenic overgrowth in wing discs carrying snr1-depletion by tissue-specific Gal4 drivers. Supplementary figure 3: Coexpression of rpr and p35 does not induce neoplastic tumorigenic overgrowth in the wing disc. Supplementary figure 4: Wing discs with snr1 loss shows neoplastic overgrowth. Supplementary figure 5: Tumorigenic overgrowth in the wing disc with mosaic clones of snr1 mutation. Supplementary figure 6: Depletion of brm or osa causes apoptotic phenotype in wing pouch region. Supplementary figure 7: Subcellular localization in salivary gland cells. Supplementary figure 8: Knockdown of other components of the SWI/SNF complex does not affect trafficking signaling. Supplementary figure 9: Transmembrane proteins are not accumulated in brm- or osa-depleted cells. Supplementary figure 10: Notch signaling is not upregulated in brm- or osa-RNAi cells. Supplementary figure 11: Depletion of brm or osa does not change JAK-STAT signaling activity. Supplementary figure 12: JNK signaling is barely affected in brm- or osa-depleted cells. Supplementary figure 13: Knockdown of dilp8 or mmp1 does not suppress snr1 depletion-induced tumorigenic overgrowth.

<p>Supplementary file 1</p>

Apicobasal cell polarity loss is a founding event in epithelial–mesenchymal transition and epithelial tumorigenesis, yet how pathological polarity loss links to plasticity remains largely unknown. To understand the mechanisms and mediators regulating plasticity upon polarity loss, we performed single-cell RNA sequencing of Drosophila ovaries, where inducing polarity-gene l(2)gl-knockdown (Lgl-KD) causes invasive multilayering of the follicular epithelia. Analyzing the integrated Lgl-KD and wildtype transcriptomes, we discovered the cells specific to the various discernible phenotypes and characterized the underlying gene expression. A genetic requirement of Keap1-Nrf2 signaling in promoting multilayer formation of Lgl-KD cells was further identified. Ectopic expression of Keap1 increased the volume of delaminated follicle cells that showed enhanced invasive behavior with significant changes to the cytoskeleton. Overall, our findings describe the comprehensive transcriptome of cells within the follicle cell tumor model at the single-cell resolution and identify a previously unappreciated link between Keap1-Nrf2 signaling and cell plasticity at early tumorigenesis.

ABSTRACT Apicobasal cell-polarity loss is a founding event in Epithelial-Mesenchymal Transition (EMT) and epithelial tumorigenesis, yet how pathological polarity loss induces plasticity changes remains largely unknown. To understand the mechanisms and mediators regulating plasticity upon polarity loss, we performed single-cell (sc) RNA sequencing of Drosophila ovaries, where inducing polarity-gene l(2)gl knockdown (Lgl-KD) causes invasive delamination of the follicular epithelia. Integrating Lgl-KD with the corresponding wild-type sc-transcriptome, we discovered clusters specific to various discernible phenotype-specific cell types and further characterized the regulons active in those cells. A genetic requirement of Keap1-Nrf2 signaling in promoting multilayer formation of Lgl-KD cells was further identified. Elevated expression of Keap1 increased the volume of delaminated follicle cells that undergo enhanced collective invasion via cytoskeletal remodeling. Overall, our findings describe the comprehensive transcriptome of the follicle-cell tumor model at the single-cell resolution and identify a previously unappreciated link between stress signaling and cell plasticity in early tumorigenesis.

Many adult tissues and organs including the intestine rely on resident stem cells to maintain homeostasis and regeneration. In mammals, the progenies of intestinal stem cells (ISCs) can dedifferentiate to generate ISCs upon ablation of resident stem cells. However, whether and how mature tissue cells generate ISCs under physiological conditions remains unknown. Here, we show that infection of the Drosophila melanogaster intestine with pathogenic bacteria induces entry of enteroblasts (EBs), which are ISC progenies, into the mitotic cycle through upregulation of epidermal growth factor receptor (EGFR)-Ras signaling. We also show that ectopic activation of EGFR-Ras signaling in EBs is sufficient to drive enteroblast mitosis cell autonomously. Furthermore, we find that the dividing enteroblasts do not gain ISC identity as a prerequisite to divide, and the regenerative ISCs are produced through EB mitosis. Taken together, our work uncovers a new role for EGFR-Ras signaling in driving EB mitosis and replenishing the ISC pool during fly intestinal regeneration, which may have important implications for tissue homeostasis and tumorigenesis in vertebrates.

Epithelial cells form continuous membranous structures for organ formation, and these cells are classified into three major morphological categories: cuboidal, columnar, and squamous. It is crucial that cells transition between these shapes during the morphogenetic events of organogenesis, yet this process remains poorly understood. All three epithelial cell shapes can be found in the follicular epithelium of Drosophila egg chamber during oogenesis. Squamous cells (SCs) are initially restricted to the anterior terminus in cuboidal shape. They then rapidly become flattened to assume squamous shape by stretching and expansion in 12 ​h during midoogenesis. Previously, we reported that Notch signaling activated a zinc-finger transcription factor Broad (Br) at the end of early oogenesis. Here we report that ecdysone and JAK/STAT pathways subsequently converge on Br to serve as an important spatiotemporal regulator of this dramatic morphological change of SCs. The early uniform pattern of Br in the follicular epithelium is directly established by Notch signaling at stage 5 of oogenesis. Later, ecdysone and JAK/STAT signaling activities synergize to suppress Br in SCs from stage 8 to 10a, contributing to proper SC squamous shape. During this process, ecdysone signaling is essential for SC stretching, while JAK/STAT regulates SC clustering and cell fate determination. This study reveals an inhibitory role of ecdysone signaling in suppressing Br in epithelial cell remodeling. In this study we also used single-cell RNA sequencing data to highlight the shift in gene expression which occurs as Br is suppressed and cells become flattened.

Ploidy variation is a cancer hallmark and is frequently associated with poor prognosis in high-grade cancers. Using a Drosophila solid-tumor model where oncogenic Notch drives tumorigenesis in a transition-zone microenvironment in the salivary gland imaginal ring, we find that the tumor-initiating cells normally undergo endoreplication to become polyploid. Upregulation of Notch signaling, however, induces these polyploid transition-zone cells to re-enter mitosis and undergo tumorigenesis. Growth and progression of the transition-zone tumor are fueled by a combination of polyploid mitosis, endoreplication, and depolyploidization. Both polyploid mitosis and depolyploidization are error prone, resulting in chromosomal copy-number variation and polyaneuploidy. Comparative RNA-seq and epistasis analysis reveal that the DNA-damage response genes, also active during meiosis, are upregulated in these tumors and are required for the ploidy-reduction division. Together, these findings suggest that polyploidy and associated cell-cycle variants are critical for increased tumor-cell heterogeneity and genome instability during cancer progression.

Epithelia form protective permeability barriers that selectively allow the exchange of material while maintaining tissue integrity under extreme mechanical, chemical, and bacterial loads. Here, we report in the Drosophila follicular epithelium a developmentally regulated and evolutionarily conserved process “patency”, wherein a breach is created in the epithelium at tricellular contacts during mid-vitellogenesis. In Drosophila, patency exhibits a strict temporal range potentially delimited by the transcription factor Tramtrack69 and a spatial pattern influenced by the dorsal-anterior signals of the follicular epithelium. Crucial for growth and lipid uptake by the oocyte, patency is also exploited by endosymbionts such as Spiroplasma pulsonii. Our findings reveal an evolutionarily conserved and developmentally regulated non-typical epithelial function in a classic model system.

Many adult tissues and organs including the intestine rely on resident stem cells to maintain homeostasis. In mammalian intestines, upon ablation of resident stem cells, the progenies of intestinal stem cells (ISCs) such as secretory cells and tuft cells can dedifferentiate to generate ISCs to drive epithelial regeneration, but whether and how the ISC progenies dedifferentiate to generate ISCs under physiological conditions remains unknown. Here we show that infection of pathogenic bacteria induces enteroblasts (EBs) as one type of ISC progenies to re-enter the mitotic cycle in the Drosophila intestine. The re-entry into mitosis is dependent on epithermal growth factor receptor (EGFR)-Ras signaling and ectopic activation of EGFR-Ras signaling in EBs is sufficient to drive EBs cell-autonomously to re-enter into mitosis. In addition, we examined whether EBs gain ISC identity as a prerequisite to divide, but the immunostaining with stem cell marker Delta shows that these dividing EBs do not gain ISC identity. After employing lineage tracing experiments, we further demonstrate that EBs dedifferentiate to generate functional ISCs after symmetric divisions of EBs. Together, our study in Drosophila intestines uncovers a new role of EGFR-Ras signaling in regulating re-entry into mitosis and dedifferentiation during regeneration and reveals a novel mechanism by which ISC progenies undergo dedifferentiation through a mitotic division, which has important implication to mammalian tissue homeostasis and tumorigenesis.