EGFR signaling controls directionality of epithelial multilayer formation upon loss of cell polarity.

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.

Damage-induced regeneration of the intestinal stem cell pool through enteroblast mitosis in the Drosophila midgut.

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.

Polyploidy in development and tumor models in Drosophila.

Polyploidy, a cell status defined as more than two sets of genomic DNA, is a conserved strategy across species that can increase cell size and biosynthetic production, but the functional aspects of polyploidy are nuanced and vary across cell types. Throughout Drosophila developmental stages (embryo, larva, pupa and adult), polyploid cells are present in numerous organs and help orchestrate development while contributing to normal growth, well-being and homeostasis of the organism. Conversely, increasing evidence has shown that polyploid cells are prevalent in Drosophila tumors and play important roles in tumor growth and invasiveness. Here, we summarize the genes and pathways involved in polyploidy during normal and tumorigenic development, the mechanisms underlying polyploidization, and the functional aspects of polyploidy in development, homeostasis and tumorigenesis in the Drosophila model.

Developmental regulation of epithelial cell cuboidal-to-squamous transition in Drosophila follicle cells.

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 â€‹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.

A single-cell atlas of adult Drosophila ovary identifies transcriptional programs and somatic cell lineage regulating oogenesis.

Oogenesis is a complex developmental process that involves spatiotemporally regulated coordination between the germline and supporting, somatic cell populations. This process has been modeled extensively using the Drosophila ovary. Although different ovarian cell types have been identified through traditional means, the large-scale expression profiles underlying each cell type remain unknown. Using single-cell RNA sequencing technology, we have built a transcriptomic data set for the adult Drosophila ovary and connected tissues. Using this data set, we identified the transcriptional trajectory of the entire follicle-cell population over the course of their development from stem cells to the oogenesis-to-ovulation transition. We further identify expression patterns during essential developmental events that take place in somatic and germline cell types such as differentiation, cell-cycle switching, migration, symmetry breaking, nurse-cell engulfment, egg-shell formation, and corpus luteum signaling. Extensive experimental validation of unique expression patterns in both ovarian and nearby, nonovarian cells also led to the identification of many new cell type-and stage-specific markers. The inclusion of several nearby tissue types in this data set also led to our identification of functional convergence in expression between distantly related cell types such as the immune-related genes that were similarly expressed in immune cells (hemocytes) and ovarian somatic cells (stretched cells) during their brief phagocytic role in nurse-cell engulfment. Taken together, these findings provide new insight into the temporal regulation of genes in a cell-type specific manner during oogenesis and begin to reveal the relatedness in expression between cell and tissues types.

A comprehensive in vivo screen for anti-apoptotic miRNAs indicates broad capacities for oncogenic synergy.

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 comprehensive 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.

CRL4Mahj E3 ubiquitin ligase promotes neural stem cell reactivation.

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.

The Hox proteins Ubx and AbdA collaborate with the transcription pausing factor M1BP to regulate gene transcription.

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.

Inhibition of Notch signaling by the p105 and p180 subunits of Drosophila chromatin assembly factor 1 is required for follicle cell proliferation.

Chromatin assembly factor 1 (CAF1), a histone chaperone that mediates the deposition of histone H3/H4 onto newly synthesized DNA, is involved in Notch signaling activation during Drosophila wing imaginal disc development. Here, we report another side of CAF1, wherein the subunits CAF1-p105 and CAF1-p180 (also known as CAF1-105 and CAF1-180, respectively) inhibit expression of Notch target genes and show this is required for proliferation of Drosophila ovarian follicle cells. Loss-of-function of either CAF1-p105 or CAF1-p180 caused premature activation of Notch signaling reporters and early expression of the Notch target Hindsight (Hnt, also known as Pebbled), leading to Cut downregulation and inhibition of follicle cell mitosis. Our studies further show Notch is functionally responsible for these phenotypes observed in both the CAF1-p105- and CAF1-p180-deficient follicle cells. Moreover, we reveal that CAF1-p105- and CAF1-p180-dependent Cut expression is essential for inhibiting Hnt expression in follicle cells during their mitotic stage. These findings together indicate a novel negative-feedback regulatory loop between Cut and Hnt underlying CAF1-p105 and CAF-p180 regulation, which is crucial for follicle cell differentiation. In conclusion, our studies suggest CAF1 plays a dual role to sustain cell proliferation by positively or negatively regulating Drosophila Notch signaling in a tissue-context-dependent manner.

Epithelial Tumors Originate in Tumor Hotspots, a Tissue-Intrinsic Microenvironment.

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.

Drosophila Model in Cancer: An Introduction.

Cancer is a cumulative manifestation of several complicated disease states that affect multiple organs. Over the last few decades, the fruit fly Drosophila melanogaster, has become a successful model for studying human cancers. The genetic simplicity and vast arsenal of genetic tools available in Drosophila provides a unique opportunity to address questions regarding cancer initiation and progression that would be extremely challenging in other model systems. In this chapter we provide a historical overview of Drosophila as a model organism for cancer research, summarize the multitude of genetic tools available, offer a brief comparison between different model organisms and cell culture platforms used in cancer studies and briefly discuss some of the latest models and concepts in recent Drosophila cancer research.

RNA helicase Belle/DDX3 regulates transgene expression in Drosophila.

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 Dicer-1 (Dcr-1) also alleviates 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 show that genetic inhibition of Bel function leads to 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.

E(y)1/TAF9 mediates the transcriptional output of Notch signaling in Drosophila.

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.

The microRNA miR-7 regulates Tramtrack69 in a developmental switch in Drosophila follicle cells.

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.

CAF-1 promotes Notch signaling through epigenetic control of target gene expression during Drosophila development.

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)mß. 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.

Histone chaperone CAF-1: essential roles in multi-cellular organism development.

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.

Regulation of broad by the Notch pathway affects timing of follicle cell development.

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.

Efficient EGFR signaling and dorsal-ventral axis patterning requires syntaxin dependent Gurken trafficking.

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.

The microRNA pathway regulates the temporal pattern of Notch signaling in Drosophila follicle cells.

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.

Topoisomerase I is an Evolutionarily Conserved Key Regulator for Satellite DNA Transcription.

Repetitive satellite DNAs, divergent in nucleic-acid sequence and size across eukaryotes, provide a physical site for centromere assembly to orchestrate chromosome segregation during the cell cycle. These non-coding DNAs are transcribed by RNA polymerase (RNAP) II and the transcription has been shown to play a role in chromosome segregation, but a little is known about the regulation of centromeric transcription, especially in higher organisms with tandemly-repeated-DNA-sequence centromeres. Using RNA interference knockdown, chemical inhibition and AID/IAA degradation, we show that Topoisomerase I (TopI), not TopII, promotes the transcription of α-satellite DNAs, the main type of satellite on centromeres in human cells. Mechanistically, TopI localizes to centromeres, binds RNAP II and facilitates RNAP II elongation on centromeres. Interestingly, in response to DNA double-stranded breaks (DSBs) induced by chemotherapy drugs or CRSPR/Cas9, α-satellite transcription is dramatically stimulated in a DNA damage checkpoint-independent but TopI-dependent manner. These DSB-induced α-satellite RNAs were predominantly derived from the α-satellite high-order repeats of human centromeres and forms into strong speckles in the nucleus. Remarkably, TopI-dependent satellite transcription also exists in mouse 3T3 and Drosophila S2 cells and in Drosophila larval imaginal wing discs and tumor tissues. Altogether, our findings herein reveal an evolutionally conserved mechanism with TopI as a key player for the regulation of satellite transcription at both cellular and animal levels.