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microRNAs (miRNAs) are ~21–22 nucleotide (nt) RNAs that mediate broad post-transcriptional regulatory networks. However, genetic analyses have shown that the phenotypic consequences of deleting individual miRNAs are generally far less overt compared to their misexpression. This suggests that miRNA deregulation may have broader phenotypic impacts during disease situations. We explored this concept in the Drosophila eye, by screening for miRNAs whose misexpression could modify the activity of pro-apoptotic factors. Via unbiased and comprehensively in vivo phenotypic assays, we identify an unexpectedly large set of miRNA hits that can suppress the action of pro-apoptotic genes hid and grim. We utilize secondary assays to validate that a subset of these miRNAs can inhibit irradiation-induced cell death. Since cancer cells might seek to evade apoptosis pathways, we modeled this situation by asking whether activation of anti-apoptotic miRNAs could serve as “second hits”. Indeed, while clones of the lethal giant larvae (lgl) tumor suppressor are normally eliminated during larval development, we find that diverse anti-apoptotic miRNAs mediate the survival of lgl mutant clones in third instar larvae. Notably, while certain anti-apoptotic miRNAs can target apoptotic factors, most of our screen hits lack obvious targets in the core apoptosis machinery. These data highlight how a genetic approach can reveal distinct and powerful activities of miRNAs in vivo, including unexpected functional synergies during disease or cancer-relevant settings.

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 twelve hours 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 the 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.

Tissue integrity and homeostasis are accomplished through strict spatial and temporal regulation of cell growth and proliferation during development. Various signaling pathways have emerged as major growth regulators across metazoans; yet, how differential growth within a tissue is spatiotemporally coordinated remains largely unclear. Here, we report a role of a growth modulator Yorkie (Yki), the Drosophila homolog of Yes-associated protein (YAP), that differentially regulates its targets in Drosophila wing imaginal discs; whereby Yki interacts with its transcriptional partner, Scalloped (Sd), the homolog of the TEAD/TEF family transcription factor in mammals, to control an essential cell cycle regulator Cyclin E (CycE). Interestingly, when Yki was coexpressed with Fizzy-related (Fzr), a Drosophila endocycle inducer and homolog of Cdh1 in mammals, surrounding hinge cells displayed larger nuclear size than distal pouch cells. The observed size difference is attributable to differential regulation of CycE, a target of Yki and Sd, the latter of which can directly bind to CycE regulatory sequences, and is expressed only in the pouch region of the wing disc starting from the late second-instar larval stage. During earlier stages of larval development, when Sd expression was not detected in the wing disc, coexpression of Fzr and Yki did not cause size differences between cells along the proximal–distal axis of the disc. We show that ectopic CycE promoted cell proliferation and apoptosis, and inhibited transcriptional activity of Yki targets. These findings suggest that spatiotemporal expression of transcription factor Sd induces differential growth regulation by Yki during wing disc development, highlighting coordination between Yki and CycE to control growth and maintain homeostasis.

The ability of neural stem cells (NSCs) to transit between quiescence and proliferation is crucial for brain development and homeostasis. Drosophila Hippo pathway maintains NSC quiescence, but its regulation during brain development remains unknown. Here, we show that CRL4Mahj, an evolutionarily conserved E3 ubiquitin ligase, is essential for NSC reactivation (exit from quiescence). We demonstrate that damaged DNA-binding protein 1 (DDB1) and Cullin4, two core components of Cullin4-RING ligase (CRL4), are intrinsically required for NSC reactivation. We have identified a substrate receptor of CRL4, Mahjong (Mahj), which is necessary and sufficient for NSC reactivation. Moreover, we show that CRL4Mahj forms a protein complex with Warts (Wts/large tumor suppressor [Lats]), a kinase of the Hippo signaling pathway, and Mahj promotes the ubiquitination of Wts. Our genetic analyses further support the conclusion that CRL4Mahj triggers NSC reactivation by inhibition of Wts. Given that Cullin4B mutations cause mental retardation and cerebral malformation, similar regulatory mechanisms may be applied to the human brain., During the transition from quiescence to reactivation of neural stem cells, the E3 ubiquitin ligase CRL4Mahj promotes their reactivation by inhibiting Wts, a core kinase of Hippo signalling pathway.

Summary 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., Graphical abstract, Highlights • Drosophila FE exhibits patency, creating transient channels across the monolayer • Patency aids in transepithelial transport of lipids for normal oocyte growth • Ttk69 regulates the temporal range of patency • EGFR and Dpp patterning pathways influence the specific spatial pattern of patency, Molecular Biology; Cell Biology; Developmental Biology

One of the most extensively used techniques inDrosophilais the Gal4/UAS binary system, which allows tissue-specific misexpression or knockdown of specific genes of interest. The original UAS vector, UASt, can only be activated for transgene expression in somatic tissues and not in the germline cells. Rørth (1998) generated UASp, a modified UAS vector that is responsive to Gal4 in both somatic and germline tissues, by replacing both thehsp70promoter and the SV40 3’UTR with the P transposase promoter and the K10 3’UTR respectively. At present, the mechanisms by which UASt is silenced in germline cells are not fully understood. Here, we report that the piRNA pathway is involved in suppressing UASt expression in ovarian germline cells. Individually knocking down or mutating components of the piRNA biogenesis pathway (e.g., Piwi, AGO3, Aub, Spn-E, and Vasa) resulted in the expression of the UASt-reporter (GFP or RFP) in the germline. An RNA-seq analysis of small RNAs revealed that thehsp70promoter of UASt is targeted by piRNAs, and in theaubmutant ovary, the amount of piRNAs targeting thehsp70promoter is reduced by around 40 folds. In contrast, the SV40 3’UTR of the UASt, which happens to be targeted by the Nonsense-mediated RNA decay (NMD) pathway, is not responsible for germline UASt suppression, as UASt-reporters with NMD-insensitive 3’UTRs fail to show germline expression. Taken together, our studies reveal a crucial role of the piRNA pathway, potentially via the suppression of thehsp70promoter, in germline UASt silencing inDrosophilaovaries.

Deregulation of the evolutionarily conserved Hippo pathway has been implicated in abnormal development of animals and in several types of cancer. One mechanism of Hippo pathway regulation is achieved by controlling the stability of its regulatory components. However, the executive E3 ligases that are involved in this process, and how the process is regulated, remain poorly defined. In this study, we identify, through a genetic candidate screen, the SCF(Slmb) E3 ligase as a novel negative regulator of the Hippo pathway in Drosophila imaginal tissues via mediation of the degradation of Expanded (Ex). Mechanistic study shows that Slmb-mediated degradation of Ex is inhibited by the Hippo signaling. Considering the fact that Hippo signaling suppresses the transcription of ex, we propose that the Hippo pathway employs a double security mechanism to ensure fine-tuned homeostasis during development.

International audience; In metazoans, the pausing of RNA polymerase II at the promoter (paused Pol II) has emerged as a widespread and conserved mechanism in the regulation of gene transcription. While critical in recruiting Pol II to the promoter, the role transcription factors play in transitioning paused Pol II into productive Pol II is, however, little known. By studying how Drosophila Hox transcription factors control transcription, we uncovered a molecular mechanism that increases productive transcription. We found that the Hox proteins AbdA and Ubx target gene promoters previously bound by the transcription pausing factor M1BP, containing paused Pol II and enriched with promoter-proximal Polycomb Group (PcG) proteins, yet lacking the classical H3K27me3 PcG signature. We found that AbdA binding to M1BP-regulated genes results in reduction in PcG binding, the release of paused Pol II, increases in promoter H3K4me3 histone marks and increased gene transcription. Linking transcription factors, PcG proteins and paused Pol II states, these data identify a two-step mechanism of Hox-driven transcription, with M1BP binding leading to Pol II recruitment followed by AbdA targeting, which results in a change in the chromatin landscape and enhanced transcription.

More and more studies have shown chromatin remodelers and histone modifiers play essential roles in regulating developmental patterns by organizing specific chromosomal architecture to establish programmed transcriptional profiles, with implications that histone chaperones execute a coordinating role in these processes. Chromatin assembly factor-1 (CAF-1), an evolutionarily conserved three-subunit protein complex, was identified as a histone chaperone coupled with DNA replication and repair in cultured mammalian cells and yeasts. Interestingly, recent findings indicate CAF-1 may have important regulatory roles during development by interacting with specific transcription factors and epigenetic regulators. In this review, we focus on the essential roles of CAF-1 in regulating heterochromatin organization, asymmetric cell division, and specific signal transduction through epigenetic modulations of the chromatin. In the end, we aim at providing a current image of facets of CAF-1 as a histone chaperone to orchestrate cell proliferation and differentiation during multi-cellular organism development.

Vesicle trafficking plays a crucial role in the establishment of cell polarity in various cellular contexts, including axis-pattern formation in the developing egg chamber of Drosophila. The EGFR ligand, Gurken (Grk), is first localized at the posterior of young oocytes for anterior–posterior axis formation and later in the dorsal anterior region for induction of the dorsal–ventral (DV) axis, but regulation of Grk localization by membrane trafficking in the oocyte remains poorly understood. Here, we report that Syntaxin 1A (Syx1A) is required for efficient trafficking of Grk protein for DV patterning. We show that Syx1A is associated with the Golgi membrane and is required for the transportation of Grk-containing vesicles along the microtubules to their dorsal anterior destination in the oocyte. Our studies reveal that the Syx1A dependent trafficking of Grk protein is required for efficient EGFR signaling during DV patterning.

Malignant tumors are caused by uncontrolled proliferation of transformed mutant cells that have lost the ability to maintain tissue integrity. Although a number of causative genetic backgrounds for tumor development have been discovered, the initial steps mutant cells take to escape tissue integrity and trigger tumorigenesis remain elusive. Here, we show through analysis of conserved neoplastic tumor-suppressor genes (nTSGs) in Drosophila wing imaginal disc epithelia that tumor initiation depends on tissue-intrinsic local cytoarchitectures, causing tumors to consistently originate in a specific region of the tissue. In this “tumor hotspot” where cells constitute a network of robust structures on their basal side, nTSG-deficient cells delaminate from the apical side of the epithelium and begin tumorigenic overgrowth by exploiting endogenous Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling activity. Conversely, in other regions, the “tumor coldspot” nTSG-deficient cells are extruded toward the basal side and undergo apoptosis. When the direction of delamination is reversed through suppression of RhoGEF2, an activator of the Rho family small GTPases, and JAK/STAT is activated ectopically in these coldspot nTSG-deficient cells, tumorigenesis is induced. These data indicate that two independent processes, apical delamination and JAK/STAT activation, are concurrently required for the initiation of nTSG-deficient-induced tumorigenesis. Given the conservation of the epithelial cytoarchitecture, tumorigenesis may be generally initiated from tumor hotspots by a similar mechanism., A genetic study in Drosophila reveals a mechanism of tumorigenesis by which pro-tumor cells initiate dysplastic tumor growth at specific "hotspot" locations in epithelial tissues., Author Summary Transformed mutant cells (pro-tumor cells) can evolve through a multistep process in which they become tumorigenic and invasive. Many genes that are involved in the different steps towards cancer development have been identified; however, how certain mutant cells destroy normal tissue organization and undergo uncontrolled proliferation during the initial stages of this process remains largely unclear. Using the epithelial tissue of the wing imaginal discs of the fruit fly (Drosophila melanogaster) larvae as a model system, we have analyzed these initial stages of inducing tumors by depletion of a neoplastic tumor suppressor gene (nTSG). We discovered that these tumors always originate from specific regions of the epithelial tissue of the wing disc. We show that in other regions that we dubbed “tumor coldspots” and that lack specific cellular structures, pro-tumor cells are eliminated from the epithelial tissue by the surrounding cells. However, in “tumor hotspots,” cells constitute specific structures in their basal side, and we found that pro-tumor cells successfully avoid potential elimination and deviate from the apical side of the tissue, initiating tumorous overgrowth. Our findings reveal the molecular and cellular mechanisms underlying the initial steps of tumorigenesis at tumor hotspots in the Drosophila imaginal wing discs.

Belle (Bel), the Drosophila homolog of the yeast DEAD-box RNA helicase DED1 and human DDX3, has been shown to be required for oogenesis and female fertility. Here we report a novel role of Bel in regulating the expression of transgenes. Abrogation of Bel by mutations or RNAi induces silencing of a variety of P-element-derived transgenes. This silencing effect depends on downregulation of their RNA levels. Our genetic studies have revealed that the RNA helicase Spindle-E (Spn-E), a nuage RNA helicase that plays a crucial role in regulating RNA processing and PIWI-interacting RNA (piRNA) biogenesis in germline cells, is required for loss-of-bel-induced transgene silencing. Conversely, Bel abrogation alleviates the nuage-protein mislocalization phenotype in spn-E mutants, suggesting a competitive relationship between these two RNA helicases. Additionally, disruption of the chromatin remodeling factor Mod(mdg4) or the microRNA biogenesis enzyme Dcr-1 also rescued the transgene-silencing phenotypes in bel mutants, suggesting the involvement of chromatin remodeling and microRNA biogenesis in loss-of-bel-induced transgene silencing. Finally we showed that genetic inhibition of Bel function led to the de novo generation of piRNAs from the transgene region inserted in the genome, suggesting a potential piRNA-dependent mechanism that may mediate transgene silencing as Bel function is inhibited. Our findings have demonstrated a novel involvement of Bel in regulating transgene expression and its loss triggers a transgene silencing mechanism mediated by protein regulators implicated in RNA processing, piRNA biogenesis, chromatin remodeling and the microRNA pathway.

Cell competition is a struggle for existence between cells in heterogeneous tissues of multicellular organisms. Loser cells, which die during cell competition, are normally viable when grown only with other loser cells, but when mixed with winner cells, they are at a growth disadvantage and undergo apoptosis. Intriguingly, several recent studies have revealed that cells bearing mutant tumor-suppressor genes, which show overgrowth and tumorigenesis in a homotypic situation, are frequently eliminated, through cell competition, from tissues in which they are surrounded by wild-type cells. Here, we focus on the regulation of cellular competitiveness and the mechanism of cell competition as inferred from two different categories of mutant cells: (1) slower-growing cells and (2) structurally defective cells. We also discuss the possible role of cell competition as an intrinsic homeostasis system through which normal cells sense and remove aberrant cells, such as precancerous cells, to maintain the integrity and normal development of tissues and organs.

SummaryThe Hippo signaling pathway regulates organ size and tissue homeostasis from Drosophila to mammals. Central to this pathway is a kinase cascade wherein Hippo (Hpo), in complex with Salvador (Sav), phosphorylates and activates Warts (Wts), which in turn phosphorylates and inactivates the Yorkie (Yki) oncoprotein, known as the YAP coactivator in mammalian cells. The FERM domain proteins Merlin (Mer) and Expanded (Ex) are upstream components that regulate Hpo activity through unknown mechanisms. Here we identify Kibra as another upstream component of the Hippo signaling pathway. We show that Kibra functions together with Mer and Ex in a protein complex localized to the apical domain of epithelial cells, and that this protein complex regulates the Hippo kinase cascade via direct binding to Hpo and Sav. These results shed light on the mechanism of Ex and Mer function and implicate Kibra as a potential tumor suppressor with relevance to neurofibromatosis.

The developmental signals that regulate the switch from genome-wide DNA replication to site-specific amplification remain largely unknown. Drosophila melanogaster epithelial follicle cells, which begin synchronized chorion gene amplification after three rounds of endocycle, provide an excellent model for study of the endocycle/gene amplification (E/A) switch. Here, we report that down-regulation of Notch signaling and activation of ecdysone receptor (EcR) are required for the E/A switch in these cells. Extended Notch activity suppresses EcR activation and prevents exit from the endocycle. Tramtrack (Ttk), a zinc-finger protein essential for the switch, is regulated negatively by Notch and positively by EcR. Ttk overexpression stops endoreplication prematurely and alleviates the endocycle exit defect caused by extended Notch activity or removal of EcR function. Our results reveal a developmental pathway that includes down-regulation of Notch, activation of the EcR, up-regulation of Ttk to execute the E/A switch, and, for the first time, the genetic interaction between Notch and ecdysone signaling in regulation of cell cycle programs and differentiation.

Metazoan development requires coordination of signaling pathways to regulate patterns of gene expression. In Drosophila, the wing imaginal disc provides an excellent model for the study of how signaling pathways interact to regulate pattern formation. The determination of the dorsal/ventral (DV) boundary of the wing disc depends on the Notch pathway, which is activated along the DV boundary and induces the expression of the homeobox transcription factor, Cut. Here, we show that Broad (Br), a zinc-finger transcription factor, is also involved in regulating Cut expression in the DV boundary region. However, Br expression is not regulated by Notch signaling in wing discs, rather, ecdysone signaling is the upstream signal that induces Br for Cut upregulation. Also, we find that the ecdysone-Br cascade upregulates cut-lacZ expression, a reporter containing a 2.7 kb cut enhancer region, implying that ecdysone signaling, similar to Notch, regulates cut at the transcriptional level. Collectively, our findings reveal that the Notch and ecdysone signaling pathways synergistically regulate Cut expression for proper DV boundary formation in the wing disc. Additionally, we show br promotes Delta, a Notch ligand, near the DV boundary to suppress aberrant high Notch activity, indicating further interaction between the two pathways for DV patterning of the wing disc.

The study of Drosophila oogenesis provides invaluable information about signaling pathway regulation and cell cycle programming. During Drosophila oogenesis, a string of egg chambers in each ovariole progressively develops toward maturity. Egg chamber development consists of 14 stages. From stage 1 to stage 6 (mitotic cycle), main-body follicle cells undergo mitotic divisions. From stage 7 to stage 10a (endocycle), follicle cells cease mitosis but continue three rounds of endoreduplication. From stage 10b to stage 13 (gene amplification), instead of whole genome duplication, follicle cells selectively amplify specific genomic regions, mostly for chorion production. So far, Drosophila oogenesis is one of the most well studied model systems used to understand cell cycle switches, which furthers our knowledge about cell cycle control machinery and sheds new light on potential cancer treatments. Here, we give a brief summary of cell cycle switches, the associated signaling pathways and factors, and the detailed experimental procedures used to study the cell cycle switches.

Dystroglycan (Dg) is a widely expressed extracellular matrix (ECM) receptor required for muscle viability, synaptogenesis, basementmembrane formation and epithelial development. As an integral component of the Dystrophin-associated glycoprotein complex, Dg plays a central role in linking the ECM and the cytoskeleton. Disruption of this linkage in skeletal muscle leads to various types of muscular dystrophies. In epithelial cells, reduced expression of Dg is associated with increased invasiveness of cancer cells. We have previously shown that Dg is required for epithelial cell polarity in Drosophila,but the mechanisms of this polarizing activity and upstream/downstream components are largely unknown. Using the Drosophila follicle-cell epithelium (FCE) as a model system, we show that the ECM molecule Perlecan(Pcan) is required for maintenance of epithelial-cell polarity. Follicle cells that lack Pcan develop polarity defects similar to those of Dg mutant cells. Furthermore, Dg depends on Pcan but not on Laminin A for its localization in the basal-cell membrane, and the two proteins bind in vitro. Interestingly, the Dg form that interacts with Pcan in the FCE lacks the mucin-like domain, which is thought to be essential for Dg ligand binding activity. Finally, we describe two examples of how Dg promotes the differentiation of the basal membrane domain: (1) by recruiting/anchoring the cytoplasmic protein Dystrophin; and (2) by excluding the transmembrane protein Neurexin. We suggest that the interaction of Pcan and Dg at the basal side of the epithelium promotes basal membrane differentiation and is required for maintenance of cell polarity in the FCE.

During Drosophila mid-oogenesis, follicular epithelial cells switch from the mitotic cycle to the specialized endocycle in which the M phase is skipped. The switch, along with cell differentiation in follicle cells, is induced by Notch signaling. We show that the homeodomain gene cut functions as a linker between Notch and genes that are involved in cell-cycle progression. Cut was expressed in proliferating follicle cells but not in cells in the endocycle. Downregulation of Cut expression was controlled by the Notch pathway and was essential for follicle cells to differentiate and to enter the endocycle properly. cut-mutant follicle cells entered the endocycle and differentiated prematurely in a cell-autonomous manner. By contrast, prolonged expression of Cut caused defects in the mitotic cycle/endocycle switch. These cells continued to express an essential mitotic cyclin, Cyclin A, which is normally degraded by the Fizzy-related-APC/C ubiquitin proteosome system during the endocycle. Cut promoted Cyclin A expression by negatively regulating Fizzy-related. Our data suggest that Cut functions in regulating both cell differentiation and the cell cycle, and that downregulation of Cut by Notch contributes to the mitotic cycle/endocycle switch and cell differentiation in follicle cells.

The transmembrane protein Dystroglycan is a central element of the dystrophin-associated glycoprotein complex, which is involved in the pathogenesis of many forms of muscular dystrophy. Dystroglycan is a receptor for multiple extracellular matrix (ECM) molecules such as Laminin, agrin and perlecan, and plays a role in linking the ECM to the actin cytoskeleton;however, how these interactions are regulated and their basic cellular functions are poorly understood. Using mosaic analysis and RNAi in the model organism Drosophila melanogaster, we show that Dystroglycan is required cell-autonomously for cellular polarity in two different cell types,the epithelial cells (apicobasal polarity) and the oocyte (anteroposterior polarity). Loss of Dystroglycan function in follicle and disc epithelia results in expansion of apical markers to the basal side of cells and overexpression results in a reduced apical localization of these same markers. In Dystroglycan germline clones early oocyte polarity markers fail to be localized to the posterior, and oocyte cortical F-actin organization is abnormal. Dystroglycan is also required non-cell-autonomously to organize the planar polarity of basal actin in follicle cells, possibly by organizing the Laminin ECM. These data suggest that the primary function of Dystroglycan in oogenesis is to organize cellular polarity; and this study sets the stage for analyzing the Dystroglycan complex by using the power ofDrosophila molecular genetics.

Age-dependent ectopic fat accumulation (EFA) in animals contributes to the progression of tissue aging and diseases such as obesity, diabetes, and cancer. However, the primary causes of age-dependent EFA remain largely elusive. Here, we characterize the occurrence of age-dependent EFA in Drosophila and identify HDAC6, a cytosolic histone deacetylase, as a suppressor of EFA. Loss of HDAC6 leads to significant age-dependent EFA, lipid composition imbalance, and reduced animal longevity on a high-fat diet. The EFA and longevity phenotypes are ameliorated by a reduction of the lipid-droplet-resident protein PLIN2. We show that HDAC6 is associated physically with the chaperone protein dHsc4/Hsc70 to maintain the proteostasis of PLIN2. These findings indicate that proteostasis collapse serves as an intrinsic cue to cause age-dependent EFA. Our study suggests that manipulation of proteostasis could be an alternative approach to the treatment of age-related metabolic diseases such as obesity and diabetes.

During Drosophila oogenesis, activation of Notch signaling in the follicular epithelium (FE) around stage 6 of oogenesis is essential for entry into the endocycle and a series of other changes such as cell differentiation and migration of subsets of the follicle cells. Notch induces the expression of zinc finger protein Hindsight and suppresses homeodomain protein Cut to regulate the mitotic/endocycle (ME) switch. Here we report that broad (br), encoding a small group of zinc-finger transcription factors resulting from alternative splicing, is a transcriptional target of Notch nuclear effector Suppressor of Hairless (Su(H)). The early pattern of Br in the FE, uniformly expressed except in the polar cells, is established by Notch signaling around stage 6, through the binding of Su(H) to the br early enhancer (brE) region. Mutation of the Su(H) binding site leads to a significant reduction of brE reporter expression in follicle cells undergoing the endocycle. Chromatin immunoprecipitation results further confirm Su(H) binding to the br early enhancer. Consistent with its expression in follicle cells during midoogenesis, loss of br function results in a delayed entry into the endocycle. Our findings suggest an important role of br in the timing of follicle cell development, and its transcriptional regulation by the Notch pathway.

The Broad-Complex (BR-C) is an early ecdysone response gene that functions during metamorphosis and encodes a family of zinc-finger transcription factors. It is expressed in a dynamic pattern during oogenesis. Its late expression in the lateral-dorsal-anterior follicle cells is related to the morphogenesis of the chorionic appendages. All four zinc-finger isoforms are expressed in oogenesis, which is consistent with the abnormal appendage phenotypes resulting from their ectopic expression. We investigated the mechanism by which the BR-C affects chorion deposition by using BrdU to follow the effects of BR-C misexpression on DNA replication and in situ hybridization to ovarian mRNA to evaluate chorion gene expression. Ectopic BR-C expression leads to prolonged endoreplication and to additional amplification of genes, besides the chorion genes, at other sites in the genome. The pattern of chorion gene expression is not affected along the anterior-posterior axis, but the follicle cells at the anterior of the oocyte fail to migrate correctly in an anterior direction when BR-C is misexpressed. We conclude that the target genes of the BR-C in oogenesis include a protein essential for endoreplication and chorion gene amplification. This may provide a link between steroid hormones and the control of DNA replication during oogenesis.

The histone chaperone CAF-1 is known for its role in DNA replication-coupled histone deposition. However, loss of function causes lethality only in higher multicellular organisms such as mice and flies, but not in unicellular organisms such as yeasts, suggesting that CAF-1 has other important functions than histone deposition during animal development. Emerging evidence indicates that CAF-1 also has a role in higher order chromatin organization and heterochromatin-mediated gene expression; it remains unclear whether CAF-1 has a role in specific signaling cascades to promote gene expression during development. Here, we report that knockdown of one of the subunits of Drosophila CAF-1, dCAF-1-p105 (Caf1-105), results in phenotypes that resemble those of, and are augmented synergistically by, mutations of Notch positive regulatory pathway components. Depletion of dCAF-1-p105 leads to abrogation of cut expression and to downregulation of other Notch target genes in wing imaginal discs. dCAF-1-p105 is associated with Suppressor of Hairless [Su(H)] and regulates its binding to the enhancer region of E(spl)mβ. The association of dCAF-1-p105 with Su(H) on chromatin establishes an active local chromatin status for transcription by maintaining a high level of histone H4 acetylation. In response to induced Notch activation, dCAF-1 associates with the Notch intracellular domain to activate the expression of Notch target genes in cultured S2 cells, manifesting the role of dCAF-1 in Notch signaling. Together, our results reveal a novel epigenetic function of dCAF-1 in promoting Notch pathway activity that regulates normal Drosophila development.

Development in multicellular organisms includes both small incremental changes and major switches of cell differentiation and proliferation status. During Drosophila oogenesis, the follicular epithelial cells undergo two major developmental switches that cause global changes in the cell-cycle program. One, the switch from the endoreplication cycle to a gene-amplification phase, during which special genomic regions undergo repeated site-specific replication, is attributed to Notch downregulation, ecdysone signaling activation and upregulation of the zinc-finger protein Tramtrack69 (Ttk69). Here, we report that the microRNA miR-7 exerts an additional layer of regulation in this developmental switch by regulating Ttk69 transcripts. miR-7 recognizes the 3′ UTR of ttk69 transcripts and regulates Ttk69 expression in a dose-dependent manner. Overexpression of miR-7 effectively blocks the switch from the endocycle to gene amplification through its regulation of ttk69. miR-7 and Ttk69 also coordinate other cell differentiation events, such as vitelline membrane protein expression, that lead to the formation of the mature egg. Our studies reveal the important role miR-7 plays in developmental decision-making in association with signal-transduction pathways.

Summary In multicellular organisms, tissue integrity and organ size are maintained through removal of aberrant or damaged cells and compensatory proliferation. Little is known, however, about this homeostasis system in postmitotic tissues, where tissue-intrinsic genetic programs constrain cell division and new cells no longer arise from stem cells. Here we show that, in postmitotic Drosophila follicular epithelia, aberrant but viable cells are eliminated through cell competition, and the resulting loss of local tissue volume triggers sporadic cellular hypertrophy to repair the tissue. This "compensatory cellular hypertrophy" is implemented by acceleration of the endocycle, a variant cell cycle composed of DNA synthesis and gap phases without mitosis, dependent on activation of the insulin/IGF-like signaling pathway. These results reveal a remarkable homeostatic mechanism in postmitotic epithelia that ensures not only elimination of aberrant cells through cell competition but also proper organ-size control that involves compensatory cellular hypertrophy induced by physical parameters.

Mahjong is a novel Lethal giant larvae-binding protein that plays a vital role in cell competition in both flies and mammals., During the initial stages of carcinogenesis, transformation events occur in a single cell within an epithelial monolayer. However, it remains unknown what happens at the interface between normal and transformed epithelial cells during this process. In Drosophila, it has been recently shown that normal and transformed cells compete with each other for survival in an epithelial tissue; however the molecular mechanisms whereby “loser cells” undergo apoptosis are not clearly understood. Lgl (lethal giant larvae) is a tumor suppressor protein and plays a crucial role in oncogenesis in flies and mammals. Here we have examined the involvement of Lgl in cell competition and shown that a novel Lgl-binding protein is involved in Lgl-mediated cell competition. Using biochemical immunoprecipitation methods, we first identified Mahjong as a novel binding partner of Lgl in both flies and mammals. In Drosophila, Mahjong is an essential gene, but zygotic mahjong mutants (mahj −/−) do not have obvious patterning defects during embryonic or larval development. However, mahj −/− cells undergo apoptosis when surrounded by wild-type cells in the wing disc epithelium. Importantly, comparable phenomena also occur in Mahjong-knockdown mammalian cells; Mahjong-knockdown Madin-Darby canine kidney epithelial cells undergo apoptosis, only when surrounded by non-transformed cells. Similarly, apoptosis of lgl −/− cells is induced when they are surrounded by wild-type cells in Drosophila wing discs. Phosphorylation of the c-Jun N-terminal kinase (JNK) is increased in mahj −/− or lgl −/− mutant cells, and expression of Puckered (Puc), an inhibitor of the JNK pathway, suppresses apoptosis of these mutant cells surrounded by wild-type cells, suggesting that the JNK pathway is involved in mahj- or lgl-mediated cell competition. Finally, we have shown that overexpression of Mahj in lgl −/− cells strongly suppresses JNK activation and blocks apoptosis of lgl −/− cells in the wild-type wing disc epithelium. These data indicate that Mahjong interacts with Lgl biochemically and genetically and that Mahjong and Lgl function in the same pathway to regulate cellular competitiveness. As far as we are aware, this is the first report that cell competition can occur in a mammalian cell culture system., Author Summary Cell transformation arises from the activation of oncoproteins and/or inactivation of tumor suppressor proteins. During the initial stage of carcinogenesis, transformation occurs in a single epithelial cell that grows within an epithelial monolayer. However, it remains unclear what happens at the interface between normal and transformed epithelial cells during this process. In Drosophila, it has been shown that normal and transformed cells often compete with each other for survival in an epithelial tissue, in a process called “cell competition.” Lethal giant larvae (Lgl) is a tumor suppressor protein in flies and mammals. Using biochemical methods, we identified Mahjong as a novel binding partner of Lgl in flies and mammals. Furthermore, we have demonstrated that Mahjong is involved in cell competition in both flies and mammals. In particular, we found that canine kidney epithelial cells depleted for Mahjong undergo apoptosis, but only when surrounded by non-transformed cells. This represents the first evidence that cell competition can occur in a mammalian cell culture system. Although it is not clear at present what molecules/signaling pathways are regulated by Lgl/Mahjong during cell competition, future studies might reveal important pathway components that could be targeted therapeutically to prevent tumor cells from “winning” in their race against normal tissue cells.

Temporal and spatial regulation of cell division is critical for proper development of multicellular organisms. An important aspect of this regulation is cell-cycle arrest, which in many cell types is coupled with differentiated status. Here we report that the polar cells—a group of follicle cells differentiated early during Drosophila oogenesis—are arrested at G2 phase and can serve as a model cell type for investigation of developmental regulation of cell-cycle arrest. On examining the effects of String, a mitosis-promoting phosphatase Cdc25 homolog, and Notch signaling in polar cells, we found that misexpression of String can trigger mitosis in existing polar cells to induce extra polar cells. Normally, differentiation of the polar cells requires Notch signaling. We found that the Notch-induced extra polar cells arise through recruitment of the neighboring cells rather than promotion of proliferation, and they are also arrested at G2 phase. Notch signaling is probably involved in down-regulating String in polar cells, thus inducing the G2 cell-cycle arrest.

Specification of the anteroposterior (AP) axis in Drosophilaoocytes requires proper organization of the microtubule and actin cytoskeleton. The establishment and regulation of cytoskeletal polarity remain poorly understood, however. Here, we show important roles for the tumor suppressor Lethal (2) giant larvae (Lgl) and atypical protein kinase C (aPKC)in regulating microtubule polarity and setting up the AP axis of the oocyte. Lgl in the germline cells regulates the localization of axis-specifying morphogens. aPKC phosphorylation of Lgl restricts Lgl activity to the oocyte posterior, thereby dividing the cortex into different domains along the AP axis. Active Lgl promotes the formation of actin-rich projections at the oocyte cortex and the posterior enrichment of the serine/threonine kinase Par-1, a key step for oocyte polarization. Our studies suggest that Lgl and its phosphorylation by aPKC may form a conserved regulatory circuitry in polarization of various cell types.

Transcriptional activation of Notch signaling targets requires the formation of a ternary complex that involves the intracellular domain of the Notch receptor (NICD), DNA-binding protein Suppressor of Hairless [Su(H), RPBJ in mammals] and coactivator Mastermind (Mam). Here, we report that E(y)1/TAF9, a component of the transcription factor TFIID complex, interacts specifically with the NICD-Su(H)-Mam complex to facilitate the transcriptional output of Notch signaling. We identified E(y)1/TAF9 in a large-scale in vivo RNA interference (RNAi) screen for genes that are involved in a Notch-dependent mitotic-to-endocycle transition in Drosophila follicle cells. Knockdown of e(y)1/TAF9 displayed Notch-mutant-like phenotypes and defects in target gene and activity reporter expression in both the follicle cells and wing imaginal discs. Epistatic analyses in these two tissues indicated that E(y)1/TAF9 functions downstream of Notch cleavage. Biochemical studies in S2 cells demonstrated that E(y)1/TAF9 physically interacts with the transcriptional effectors of Notch signaling Su(H) and NICD. Taken together, our data suggest that the association of the NICD-Su(H)-Mastermind complex with E(y)1/TAF9 in response to Notch activation recruits the transcription initiation complex to induce Notch target genes, coupling Notch signaling with the transcription machinery.

In many developmental processes, polyploid cells are generated by a variation of the normal cell cycle called the endocycle in which cells increase their genomic content without dividing. How the transition from the normal mitotic cycle to endocycle is regulated is poorly understood. We show that the transition from mitotic cycle to endocycle in the Drosophila follicle cell epithelium is regulated by the Notch pathway. Loss of Notch function in follicle cells or its ligand Delta function in the underlying germline disrupts the normal transition of the follicle cells from mitotic cycle to endocycle, mitotic cycling continues, leading to overproliferation of these cells. The regulation is at the transcriptional level, as Su(H), a downstream transcription factor in the pathway, is also required cell autonomously in follicle cells for proper transitioning to the endocycle. One target of Notch and Su(H) is likely to be the G2/M cell cycle regulator String, a phosphatase that activates Cdc2 by dephosphorylation. String is normally repressed in the follicle cells just before the endocycle transition, but is expressed when Notch is inactivated. Analysis of the activity of String enhancer elements in follicle cells reveals the presence of an element that promotes expression of String until just before the onset of polyploidy in wild-type follicle cells but well beyond this stage in Notch mutant follicle cells. This suggests that it may be the target of the endocycle promoting activity of the Notch pathway. A second element that is insensitive to Notch regulation promotes String expression earlier in follicle cell development, which explains why Notch, while active at both stages, represses String only at the mitotic cycle-endocycle transition.

We have identified partial loss of function mutations in class VI unconventional myosin, 95F myosin, which results in male sterility. During spermatogenesis the germ line precursor cells undergo mitosis and meiosis to form a bundle of 64 spermatids. The spermatids remain interconnected by cytoplasmic bridges until individualization. The process of individualization involves the formation of a complex of cytoskeletal proteins and membrane, the individualization complex (IC), around the spermatid nuclei. This complex traverses the length of each spermatid resolving the shared membrane into a single membrane enclosing each spermatid. We have determined that 95F myosin is a component of the IC whose function is essential for individualization. In wild-type testes, 95F myosin localizes to the leading edge of the IC. Two independent mutations in 95F myosin reduce the amount of 95F myosin in only a subset of tissues, including the testes. This reduction of 95F myosin causes male sterility as a result of defects in spermatid individualization. Germ line transformation with the 95F myosin heavy chain cDNA rescues the male sterility phenotype. IC movement is aberrant in these 95F myosin mutants, indicating a critical role for 95F myosin in IC movement. This report is the first identification of a component of the IC other than actin. We propose that 95F myosin is a motor that participates in membrane reorganization during individualization.

We report the analysis of a number of lines of Drosophila melanogaster containing insertions of the yeast gal4 gene. By crossing a UAS-lacZ fusion gene as a reporter into these lines, we analysed the expression patterns of beta-galactosidase during oogenesis. Since there is no expression of GAL4 in the germ-line in these experiments, this is an ideal system for the analysis of expression patterns in sub-sets of follicle cells. These lines provide ideal markers for sets of follicle cells, e.g. anterior or posterior polar cells for studying genetic interactions in oogenesis; however, they can also be used in the same way as conventional enhancer traps to clone nearby genes with similar expression patterns. The advantages of this dual gal4/UAS system over conventional enhancer trapping includes the possibility of GAL4-directed misexpression and antisense expression studies to establish the function of the genes we identified during follicle cell determination and differentiation. These studies could lead to the isolation of homologous genes crucial in mammalian oogenesis. Understanding how the somatic cells and germ cells interact to promote growth and maturation of the mammalian follicle and oocyte could well be crucial for improving the fertility of eggs used for in-vitro fertilization programmes, and could provide methods for assessing the quality of eggs.

Multicellular development requires the correct spatial and temporal regulation of cell division and differentiation. These processes are frequently coordinated by the activities of various signaling pathways such as Notch signaling. From a screen for modifiers of Notch signaling in Drosophila we have identified the RNA helicase Belle, a recently described component of the RNA interference pathway, as an important regulator of the timing of Notch activity in follicle cells. We found that loss of Belle delays activation of Notch signaling, which results in delayed follicle cell differentiation and defects in the cell cycle. Because mutations in well-characterized microRNA components phenocopied the Notch defects observed in belle mutants, Belle might be functioning in the microRNA pathway in follicle cells. The effect of loss of microRNAs on Notch signaling occurs upstream of Notch cleavage, as expression of the constitutively active intracellular domain of Notch in microRNA-defective cells restored proper activation of Notch. Furthermore, we present evidence that the Notch ligand Delta is an important target of microRNA regulation in follicle cells and regulates the timing of Notch activation through cis inhibition of Notch. Here we have uncovered a complex regulatory process in which the microRNA pathway promotes Notch activation by repressing Delta-mediated inhibition of Notch in follicle cells.

The formation of an anterior–posterior (AP) gradient of microtubules in Drosophila oocytes is essential for specification of the AP axis. Proper microtubule organization in the oocyte requires the function of serine/threonine kinase Par-1. The N1S isoform of Par-1 is enriched at the posterior cortex of the oocyte from stage 7 of oogenesis. Here we report that posterior restriction of Par-1 (N1S) kinase activity is critical for microtubule AP gradient formation. Egg chambers with excessive and ectopic Par-1 (N1S) kinase activity in the germline cells display phenotypes similar to those of egg chambers treated with the microtubule-depolymerizing drug colcemid: depolymerization of microtubules in the oocyte and disruption of oocyte nucleus localization. A phosphorylation target of Par-1, the microtubule-associated protein Tau, is also involved in oocyte polarity formation, and overexpression of Tau alleviates the phenotypes caused by ectopic Par-1 (N1S) kinase activity, suggesting that Par-1 regulates oocyte polarity at least partly through Tau. Our findings reveal that maintaining proper levels of Par-1 at correct position in the oocyte is key to oocyte polarity formation and that the conserved role of Par-1 and Tau is crucial for the establishment of an AP gradient of microtubules and for AP axis specification.

Intercellular communication between the somatic and germline cells is vital to development of the Drosophila egg chamber. One critical outcome of this communication is the polarization of the oocyte along the anterior–posterior axis, a process induced by an unknown signal from the somatic follicle cells to the oocyte. The existence of this signal has been inferred from several reports demonstrating that the differentiation and patterning of the follicle cells by the spatially restricted activation of certain cell-signaling pathways is necessary for axis formation in the oocyte. These reports have also provided a framework for understanding how these signaling pathways are integrated to generate the follicle-cell pattern, but the precise role of the follicle cells in anterior–posterior axis formation remains enigmatic. Research has identified several genes that appear to be involved in the polarizing communication from the follicle cells to the oocyte. Interestingly the proteins encoded by most of these genes are associated with the extracellular matrix, suggesting a pivotal role for this complex biological component in the polarizing communication between the follicle cells and the oocyte. This review summarizes the findings in this area, and uses the experimental analyses of these genes to evaluate various models describing the possible nature of the polarizing signal, and the role of these genes in it.