Bantam microRNA Research Articles

Evaluation of microRNA variant maturation prior to genome edition

Assessment of the functionality of individual microRNA/target sites is a crucial issue. Genome editing techniques should theoretically permit a fine functional exploration of such interactions, allowing the mutation of microRNAs or individual binding sites in a complete in vivo setting, therefore abrogating or restoring individual interactions on demand. A major limitation to this experimental strategy is the influence of microRNA sequence on its accumulation level, which introduces a confounding effect when assessing phenotypic rescue by compensatorily mutated microRNA and target site. Here we describe a simple assay to identify microRNA variants most likely to accumulate at wild-type levels even though their sequence has been mutated. In this assay, quantification of a reporter construct in cultured cells predicts the efficiency of an early biogenesis step, the Drosha-dependent cleavage of microRNA precursors, which appears to be a major determinant of microRNA accumulation in our variant collection. This system allowed the generation of a mutant Drosophila strain expressing a bantam microRNA variant at wild-type levels.

The Drosophila microRNA bantam regulates excitability in adult mushroom body output neurons to promote early night sleep

SummarySleep circuitry evolved to have both dedicated and context-dependent modulatory elements. Identifying modulatory subcircuits and understanding their molecular machinery is a major challenge for the sleep field. Previously, we identified 25 sleep-regulating microRNAs in Drosophila melanogaster, including the developmentally important microRNA bantam. Here we show that bantam acts in the adult to promote early nighttime sleep through a population of glutamatergic neurons that is intimately involved in applying contextual information to behaviors, the γ5β′2a/β′2mp/β′2mp_bilateral Mushroom Body Output Neurons (MBONs). Calcium imaging revealed that bantam inhibits the activity of these cells during the early night, but not the day. Blocking synaptic transmission in these MBONs rescued the effect of bantam knockdown. This suggests bantam promotes early night sleep via inhibition of the γ5β′2a/β′2mp/β′2mp_bilateral MBONs. RNAseq identifies Kelch and CCHamide-2 receptor as possible mediators, establishing a new role for bantam as an active regulator of sleep and neural activity in the adult fly.

Yorkie drives supercompetition by non-autonomous induction of autophagy via bantam microRNA in Drosophila

Mutations in the tumor-suppressor Hippo pathway lead to activation of the transcriptional coactivator Yorkie (Yki), which enhances cell proliferation autonomously and causes cell death non-autonomously. While Yki-induced cell proliferation has extensively been studied, the mechanism by which Yki causes cell death in nearby wild-type cells, a phenomenon called supercompetition, and its role in tumorigenesis remained unknown. Here, we show that Yki-induced supercompetition is essential for tumorigenesis and is driven by non-autonomous induction of autophagy. Clones of cells mutant for a Hippo pathway component fat activate Yki and cause autonomous tumorigenesis and non-autonomous cell death in Drosophila eye-antennal discs. Through a genetic screen in Drosophila, we find that mutations in autophagy-related genes or NF-κB genes in surrounding wild-type cells block both fat-induced tumorigenesis and supercompetition. Mechanistically, fat mutant cells upregulate Yki-target microRNA bantam, which elevates protein synthesis levels via activation of TOR signaling. This induces elevation of autophagy in neighboring wild-type cells, which leads to downregulation of IκB Cactus and thus causes NF-κB-mediated induction of the cell death gene hid. Crucially, upregulation of bantam is sufficient to make cells to be supercompetitors and downregulation of endogenous bantam is sufficient for cells to become losers of cell competition. Our data indicate that cells with elevated Yki-bantam signaling cause tumorigenesis by non-autonomous induction of autophagy that kills neighboring wild-type cells.

JNK and Yorkie drive tumor malignancy by inducing L-amino acid transporter 1 in Drosophila.

Identifying a common oncogenesis pathway among tumors with different oncogenic mutations is critical for developing anti-cancer strategies. Here, we performed transcriptome analyses on two different models of Drosophila malignant tumors caused by Ras activation with cell polarity defects (RasV12/scrib-/-) or by microRNA bantam overexpression with endocytic defects (bantam/rab5-/-), followed by an RNAi screen for genes commonly essential for tumor growth and malignancy. We identified that Juvenile hormone Inducible-21 (JhI-21), a Drosophila homolog of the L-amino acid transporter 1 (LAT1), is upregulated in these malignant tumors with different oncogenic mutations and knocking down of JhI-21 strongly blocked their growth and invasion. JhI-21 expression was induced by simultaneous activation of c-Jun N-terminal kinase (JNK) and Yorkie (Yki) in these tumors and thereby contributed to tumor growth and progression by activating the mTOR-S6 pathway. Pharmacological inhibition of LAT1 activity in Drosophila larvae significantly suppressed growth of RasV12/scrib-/- tumors. Intriguingly, LAT1 inhibitory drugs did not suppress growth of bantam/rab5-/- tumors and overexpression of bantam rendered RasV12/scrib-/- tumors unresponsive to LAT1 inhibitors. Further analyses with RNA sequencing of bantam-expressing clones followed by an RNAi screen suggested that bantam induces drug resistance against LAT1 inhibitors via downregulation of the TMEM135-like gene CG31157. Our observations unveil an evolutionarily conserved role of LAT1 induction in driving Drosophila tumor malignancy and provide a powerful genetic model for studying cancer progression and drug resistance.

The Upd3 cytokine couples inflammation to maturation defects in Drosophila

Developmental transitions, such as puberty or metamorphosis, are tightly controlled by steroid hormones and can be delayed by the appearance of growth abnormalities, developmental tumors, or inflammatory disorders such as inflammatory bowel disease or cystic fibrosis.1-4 Here, we used a highly inflammatory epithelial model of malignant transformation in Drosophila5,6 to unravel the role of Upd3-a cytokine with homology to interleukin-6-and the JAK/STAT signaling pathway in coupling inflammation to a delay in metamorphosis. We present evidence that Upd3 produced by malignant and nearby cell populations signals to the prothoracic gland-an endocrine tissue primarily dedicated to the production of the steroid hormone ecdysone-to activate JAK/STAT and bantam microRNA (miRNA) and to delay metamorphosis. Upd cytokines produced by the tumor site contribute to increasing the systemic levels of Upd3 by amplifying its expression levels in a cell-autonomous manner and by inducing Upd3 expression in neighboring tissues in a non-autonomous manner, culminating in a major systemic response to prevent larvae from initiating pupa transition. Our results identify a new regulatory network impacting on ecdysone biosynthesis and provide new insights into the potential role of inflammatory cytokines and the JAK/STAT signaling pathway in coupling inflammation to delays in puberty.

The Drosophila Microrna Bantam Regulates Excitability in Adult Mushroom Body Output Neurons to Promote Early Night Sleep

SummaryThe decision to sleep requires integration of information about the internal state of the animal and its immediate external environment. Sleep circuitry therefore evolved to have both dedicated and context-dependent modulatory neuronal elements [1-3]. Identifying these important subcircuits and understanding the molecular mechanisms regulating their roles is a major challenge for the sleep field. microRNAs, non-coding RNA transcripts which posttranscriptionally control gene expression [4], have been implicated in sleep regulation [5-10]. Our previous screen [11] identified 25 sleep-regulating microRNAs in Drosophila melanogaster , including the developmentally-important [12-15] microRNA bantam ( ban ). Here we show that ban acts in the adult brain to promote early nighttime sleep through a population of glutamatergic neurons that is intimately involved in applying contextual information to behaviors [16]- the γ5β′2a/β′2mp/β′2mp_bilateral Mushroom Body Output Neurons (MBONs). GCaMP calcium imaging revealed that bantam inhibits the neural activity of these cells during the early night, but not the day. Blocking synaptic transmission in these MBONs rescued the effect of ban knockdown. Together these results suggest ban promotes early night sleep via inhibition of the γ5β′2a/β′2mp/β′2mp_bilateral MBONs. RNAseq identifies Kelch and CCHamide-2 receptor as mediators of this role. These experiments establish a new role for bantam as an active regulator of sleep and neural activity in the adult fly brain. Notably, the gene for BRS3 (Bombesin receptor subtype-3), the human ortholog of CCHa2-R, hosts an MRE for the mammalian ortholog of ban , miR-450b-3p [17-19]. The regulation of orexin neurons by this receptor [20] implies this pathway is highly conserved.

ADF1 and BEAF-32 chromatin proteins affect nucleosome positioning and DNA decompaction in 61C7/C8 interband region of Drosophila melanogaster polytene chromosomes

The formation of interphase chromosomes is a multi-level process in which DNA is compacted several thousandfold by association with histones and non-histone proteins. The first step of compaction includes the formation of nucleosomes – the basic repeating units of chromatin. Further packaging occurs due to DNA binding to histone H1 and non-histone proteins involved in enhancer-promoter and insulator interactions. Under these conditions, the genome retains its functionality due to the dynamic and uneven DNA compaction along the chromatin fiber. Since the DNA compaction level affects the transcription activity of a certain genomic region, it is important to understand the interplay between the factors acting at different levels of the packaging process. Drosophila polytene chromosomes are an excellent model system for studying the molecular mechanisms that determine DNA compaction degree. The unevenness of DNA packaging along the chromatin fiber is easily observed along these chromosomes due to their large size and specific banding pattern. The purpose of this study was to figure out the role of two non-histone regulatory proteins, ADF1 and BEAF-32, in the DNA packaging process from nucleosome positioning to the establishment of the final chromosome structure. We studied the impact of mutations that affect ADF1 and BEAF-32 binding sites on the formation of 61C7/C8 interband – one of the decompacted regions of Drosophila polytene chromosomes. We show that such mutations led to the collapse of an interband, which was accompanied with increased nucleosome stability. We also find that ADF1 and BEAF-32 binding sites are essential for the rescue of lethality caused by the null allele of bantam microRNA gene located in the region 61C7/C8.

Autophagy-Mediated Cholesterol Trafficking Controls Steroid Production

Steroid hormones are important signaling molecules that regulate growth and drive the development of many cancers. These factors act as long-range signals that systemically regulate the growth of the entire organism, whereas the Hippo/Warts tumor-suppressor pathway acts locally to limit organ growth. We show here that autophagy, a pathway that mediates the degradation of cellular components, also controls steroid production. This process is regulated by Warts (in mammals, LATS1/2) signaling, via its effector microRNA bantam, in response to nutrients. Specifically, autophagy-mediated mobilization and trafficking of the steroid precursor cholesterol from intracellular stores controls the production of the Drosophila steroid ecdysone. Furthermore, we also show that bantam regulates this process via the ecdysone receptor and Tor signaling, identifying pathways through which bantam regulates autophagy and growth. The Warts pathway thus promotes nutrient-dependent systemic growth during development by autophagy-dependent steroid hormone regulation (ASHR). These findings uncover an autophagic trafficking mechanism that regulates steroid production.

The Corazonin-PTTH Neuronal Axis Controls Systemic Body Growth by Regulating Basal Ecdysteroid Biosynthesis in  <i>Drosophila melanogaster</i>

Growth and maturation are coordinated by steroid hormone biosynthesis in various animal phyla. The insect steroid hormone, ecdysteroid, coordinates growth and maturation, represented by molting and metamorphosis. In Drosophila melanogaster, prothoracicotropic hormone (PTTH) neurons stimulate in generating peak levels of ecdysteroid to trigger maturation. In addition, recent studies have shed light on the role of PTTH signaling in basal ecdysteroid biosynthesis, which negatively affects systemic growth prior to maturation. However, it remains unclear how PTTH signaling is regulated for basal ecdysteroid biosynthesis. Here, we report that Corazonin (Crz)-producing neurons regulate basal ecdysteroid biosynthesis by affecting PTTH neurons. Inhibition of Crz neuronal activity increased pupal size, whereas it had little effect on pupariation timing. This phenotype resulted from enhanced growth and a delay in basal ecdysteroid elevation during the mid-third instar larval (L3) stage. Silencing of Crz in Crz neurons resulted in increased levels of the microRNA bantam, which represses basal ecdysteroid biosynthesis. Interestingly, Crz receptor (CrzR) expression in PTTH neurons was higher during the mid- than during the late-L3 stage. Silencing of CrzR in PTTH neurons increased pupal size, phenocopying the inhibition of Crz neuronal activity. When Crz neurons were optogenetically activated, a strong calcium response was observed in PTTH neurons in the mid-, but not the late-L3 stage. These data suggest that the Crz-PTTH neuronal axis modulates basal, but not peak ecdysteroid biosynthesis. Most significantly, this study uncovered a regulatory neuronal system affecting ecdysteroid biosynthesis in a developmental stage-specific manner.

Expressing MicroRNA Bantam Sponge Drastically Improves the Insecticidal Activity of Baculovirus via Increasing the Level of Ecdysteroid Hormone in Spodoptera exigua Larvae.

Bantam is a conserved miRNA highly expressed in insects. We previously showed that the antisense inhibitor (antagomiR) of bantam improved the infection by baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) in Spodoptera exigua and S. litura larvae. Here, we constructed a recombinant AcMNPV (vPH-banS) expressing bantam sponge, an mRNA containing eight antisense binding sites for bantam. Infection with wild type AcMNPV (WT) or the control recombinant virus vPH resulted in a significant increase of bantam level, whereas infection with vPH-banS led to an approximately 40% reduction of bantam in both Sf9 cells and S. exigua larvae. Although, comparable production of budded virus and polyhedra were detected in vPH-banS-, vPH-, and WT-infected Sf9 cells, vPH-banS showed remarkably increased insecticidal activity in S. exigua larvae. The 50% lethal concentration and the median lethal time of vPH-banS was only 1/40 and 1/2, respectively, of both vPH and WT. Further analysis showed that the level of molting hormone 20-hydroxyecdysone (20E) was significantly higher in larvae infected with vPH-banS than those infected with vPH or WT. This was confirmed by the result that the larvae treated with bantam inhibitor also had a markedly increased 20E level. Moreover, feeding larvae with 20E increased the virus-mediated mortality, whereas feeding with juvenile hormone partially reverted the high insecticidal effect of vPH-banS. Together, our results revealed that vPH-banS infection suppresses the level of bantam, and in turn elevates level of 20E in infected insects, resulting in increased susceptibility to baculovirus infection. Our study provided a novel approach to improve a baculovirus bio-insecticide by interfering with a key homeostasis-regulating miRNA of the host.

Bantam microRNA is a negative regulator of the Drosophila decapentaplegic pathway

ABSTRACTDecapentaplegic (Dpp), the Drosophila homolog of the vertebrate bone morphogenetic protein (BMP2/4), is crucial for patterning and growth in many developmental contexts. The Dpp pathway is regulated at many different levels to exquisitely control its activity. We show that bantam (ban), a microRNA, modulates Dpp signaling activity. Over expression of ban decreases phosphorylated Mothers against decapentaplegic (Mad) levels and negatively affects Dpp pathway transcriptional target genes, while null mutant clones of ban upregulate the pathway. We provide evidence that dpp upregulates ban in the wing imaginal disc, and attenuation of Dpp signaling results in a reduction of ban expression, showing that they function in a feedback loop. Furthermore, we show that this feedback loop is important for maintaining anterior-posterior compartment boundary stability in the wing disc through regulation of optomotor blind (omb), a known target of the pathway. Our results support a model that ban functions with dpp in a negative feedback loop.

The equilibrium between antagonistic signaling pathways determines the number of synapses in Drosophila.

The number of synapses is a major determinant of behavior and many neural diseases exhibit deviations in that number. However, how signaling pathways control this number is still poorly understood. Using the Drosophila larval neuromuscular junction, we show here a PI3K-dependent pathway for synaptogenesis which is functionally connected with other previously known elements including the Wit receptor, its ligand Gbb, and the MAPkinases cascade. Based on epistasis assays, we determined the functional hierarchy within the pathway. Wit seems to trigger signaling through PI3K, and Ras85D also contributes to the initiation of synaptogenesis. However, contrary to other signaling pathways, PI3K does not require Ras85D binding in the context of synaptogenesis. In addition to the MAPK cascade, Bsk/JNK undergoes regulation by Puc and Ras85D which results in a narrow range of activity of this kinase to determine normalcy of synapse number. The transcriptional readout of the synaptogenesis pathway involves the Fos/Jun complex and the repressor Cic. In addition, we identified an antagonistic pathway that uses the transcription factors Mad and Medea and the microRNA bantam to down-regulate key elements of the pro-synaptogenesis pathway. Like its counterpart, the anti-synaptogenesis signaling uses small GTPases and MAPKs including Ras64B, Ras-like-a, p38a and Licorne. Bantam downregulates the pro-synaptogenesis factors PI3K, Hiw, Ras85D and Bsk, but not AKT. AKT, however, can suppress Mad which, in conjunction with the reported suppression of Mad by Hiw, closes the mutual regulation between both pathways. Thus, the number of synapses seems to result from the balanced output from these two pathways.

Warts Signaling Controls Organ and Body Growth through Regulation of Ecdysone

Coordination of growth between individual organs and the whole body is essential during development to produce adults with appropriate size and proportions [1, 2]. How local organ-intrinsic signals and nutrient-dependent systemic factors are integrated to generate correctly proportioned organisms under different environmental conditions is poorly understood. In Drosophila, Hippo/Warts signaling functions intrinsically to regulate tissue growth and organ size [3, 4], whereas systemic growth is controlled via antagonistic interactions of the steroid hormone ecdysone and nutrient-dependent insulin/insulin-like growth factor (IGF) (insulin) signaling [2, 5]. The interplay between insulin and ecdysone signaling regulates systemic growth and controls organismal size. Here, we show that Warts (Wts; LATS1/2) signaling regulates systemic growth in Drosophila by activating basal ecdysone production, which negatively regulates body growth. Further, we provide evidence that Wts mediates effects of insulin and the neuropeptide prothoracicotropic hormone (PTTH) on regulation of ecdysone production through Yorkie (Yki; YAP/TAZ) and the microRNA bantam (ban). Thus, Wts couples insulin signaling with ecdysone production to adjust systemic growth in response to nutritional conditions during development. Inhibition of Wts activity in the ecdysone-producing cells non-autonomously slows the growth of the developing imaginal-disc tissues while simultaneously leading to overgrowth of the animal. This indicates that ecdysone, while restricting overall body growth, is limiting for growth of certain organs. Our data show that, in addition to its well-known intrinsic role in restricting organ growth, Wts/Yki/ban signaling also controls growth systemically by regulating ecdysone production, a mechanism that we propose controls growth between tissues and organismal size in response to nutrient availability.

The bantam microRNA acts through Numb to exert cell growth control and feedback regulation of Notch in tumor-forming stem cells in the Drosophila brain.

Notch (N) signaling is central to the self-renewal of neural stem cells (NSCs) and other tissue stem cells. Its deregulation compromises tissue homeostasis and contributes to tumorigenesis and other diseases. How N regulates stem cell behavior in health and disease is not well understood. Here we show that N regulates bantam (ban) microRNA to impact cell growth, a process key to NSC maintenance and particularly relied upon by tumor-forming cancer stem cells. Notch signaling directly regulates ban expression at the transcriptional level, and ban in turn feedback regulates N activity through negative regulation of the Notch inhibitor Numb. This feedback regulatory mechanism helps maintain the robustness of N signaling activity and NSC fate. Moreover, we show that a Numb-Myc axis mediates the effects of ban on nucleolar and cellular growth independently or downstream of N. Our results highlight intricate transcriptional as well as translational control mechanisms and feedback regulation in the N signaling network, with important implications for NSC biology and cancer biology.

Spalt is functionally conserved in Locusta and Drosophila to promote wing growth

Locusta has strong fly wings to ensure its long distance migration, but the molecular mechanism that regulates the Locusta wing development is poorly understood. To address the developmental mechanism of the Locusta flying wing, we cloned the Dpp target gene spalt (sal) and analyzed its function in wing growth in the Locusta. The Locusta wing size is apparently reduced with vein defects when sal is interfered by injection of dsRNA, indicating that sal is required for locust wing growth and vein formation. This function is conserved during the Drosophila wing development. To better understand sal’s function in wing growth, we then used Drosophila wing disc as a model for further study. We found that sal promotes cell proliferation in the whole wing disc via positive regulation of a microRNA bantam. Our results firstly unravel sal’s function in the Locusta wing growth and confirm a highly conserved function of sal in Locusta and Drosophila.

The Effect of MicroRNA bantam on Baculovirus AcMNPV Infection in Vitro and in Vivo.

The role of microRNA bantam, one of the most abundant microRNAs in Sf9 cells, was studied for its role in baculovirus infection in vitro and in vivo. The expression level of bantam was increased after AcMNPV infection in Sf9 cells and in Spodoptera litura larvae. In Sf9 cells, application of bantam inhibitor or mimic altered the expression of many virus genes, the most affected gene being lef8, gp41 and p10, the expression level of which was increased by 8, 10 and 40 times, respectively, in the presence of bantam inhibitor. Virus DNA replication was decreased in the presence of bantam mimic and increased in the presence of bantam inhibitor in a dose dependent manner. However, the production of budded virus did not change significantly. Feeding the larvae of S. litura and Spodoptera exigua with bantam antagomiR, a more stable form of the inhibitor, resulted in an abnormal larval growth and a decreased pupation rate. In S. litura, larvae died 3.5 days sooner than the control when bantam antagomiR was applied, together with AcMNPV. In infected S. exigua, larval mortality increased from 47% without antagomiR to 80% with it. The results suggest that microRNA bantam plays an important role in insect growth, as well as in baculovirus-insect interaction.

HDAC inhibitor misprocesses bantam oncomiRNA, but stimulates hid induced apoptotic pathway.

Apoptosis or programmed cell death is critical for embryogenesis and tissue homeostasis. Uncontrolled apoptosis leads to different human disorders including immunodeficiency, autoimmune disorder and cancer. Several small molecules that control apoptosis have been identified. Here, we have shown the functional role of triazole derivative (DCPTN-PT) that acts as a potent HDAC inhibitor and mis-express proto onco microRNA (miRNA) bantam. To further understanding the mechanism of action of the molecule in apoptotic pathway, a series of experiments were also performed in Drosophila, a well known model organism in which the nature of human apoptosis is very analogous. DCPTN-PT mis processes bantam microRNA and alters its down regulatory target hid function and cleavage of Caspase-3 which in turn influence components of the mitochondrial apoptotic pathway in Drosophila. However regulatory microRNAs in other pro-apoptotic genes are not altered. Simultaneously, treatment of same molecule also affects the mitochondrial regulatory pathway in human tumour cell lines suggesting its conservative nature between fly and human. It is reasonable to propose that triazole derivative (DCPTN-PT) controls bantam oncomiRNA and increases hid induced apoptosis and is also able to influence mitochondrial apoptotic pathway.

Tie-mediated signal from apoptotic cells protects stem cells in Drosophila melanogaster.

Many types of normal and cancer stem cells are resistant to killing by genotoxins, but the mechanism for this resistance is poorly understood. Here we show that adult stem cells in Drosophila melanogaster germline and midgut are resistant to ionizing radiation (IR) or chemically induced apoptosis and dissect the mechanism for this protection. We find that upon IR the receptor tyrosine kinase Tie/Tie-2 is activated, leading to the upregulation of microRNA bantam that represses FOXO-mediated transcription of pro-apoptotic Smac/DIABLO orthologue, Hid in germline stem cells. Knockdown of the IR-induced putative Tie ligand, Pvf1, a functional homologue of human Angiopoietin, in differentiating daughter cells renders germline stem cells sensitive to IR, suggesting that the dying daughters send a survival signal to protect their stem cells for future repopulation of the tissue. If conserved in cancer stem cells, this mechanism may provide therapeutic options for the eradication of cancer.

Control of Drosophila Type I and Type II central brain neuroblast proliferation by bantam microRNA.

Post-transcriptional regulation of stem cell self-renewal by microRNAs is emerging as an important mechanism controlling tissue homeostasis. Here, we provide evidence that bantam microRNA controls neuroblast number and proliferation in the Drosophila central brain. Bantam also supports proliferation of transit-amplifying intermediate neural progenitor cells in type II neuroblast lineages. The stem cell factors brat and prospero are identified as bantam targets acting on different aspects of these processes. Thus, bantam appears to act in multiple regulatory steps in the maintenance and proliferation of neuroblasts and their progeny to regulate growth of the central brain.

The retinal determination gene Dachshund restricts cell proliferation by limiting the activity of the Homothorax-Yorkie complex.

The Drosophila transcriptional co-activator protein Yorkie and its vertebrate orthologs YAP and TAZ are potent oncogenes, whose activity is normally kept in check by the upstream Hippo kinase module. Upon its translocation into the nucleus, Yorkie forms complexes with several tissue-specific DNA-binding partners, which help to define the tissue-specific target genes of Yorkie. In the progenitor cells of the eye imaginal disc, the DNA-binding transcription factor Homothorax is required for Yorkie-promoted proliferation and survival through regulation of the bantam microRNA (miRNA). The transit from proliferating progenitors to cell cycle quiescent precursors is associated with the progressive loss of Homothorax and gain of Dachshund, a nuclear protein related to the Sno/Ski family of co-repressors. We have identified Dachshund as an inhibitor of Homothorax-Yorkie-mediated cell proliferation. Loss of dachshund induces Yorkie-dependent tissue overgrowth. Conversely, overexpressing dachshund inhibits tissue growth, prevents Yorkie or Homothorax-mediated cell proliferation of disc epithelia and restricts the transcriptional activity of the Yorkie-Homothorax complex on the bantam enhancer in Drosophila cells. In addition, Dachshund collaborates with the Decapentaplegic receptor Thickveins to repress Homothorax and Cyclin B expression in quiescent precursors. The antagonistic roles of Homothorax and Dachshund in Yorkie activity, together with their mutual repression, ensure that progenitor and precursor cells are under distinct proliferation regimes. Based on the crucial role of the human dachshund homolog DACH1 in tumorigenesis, our work suggests that DACH1 might prevent cellular transformation by limiting the oncogenic activity of YAP and/or TAZ.