Timeless noncoding DNA contains cell-type preferential enhancers important for proper Drosophila circadian regulation.

To address the contribution of transcriptional regulation to Drosophila clock gene expression and to behavior, we generated a series of CRISPR-mediated deletions within two regions of the circadian gene timeless (tim), an intronic E-box region and an upstream E-box region that are both recognized by the key transcription factor Clock (Clk) and its heterodimeric partner Cycle. The upstream deletions but not an intronic deletion dramatically impact tim expression in fly heads; the biggest upstream deletion reduces peak RNA levels and tim RNA cycling amplitude to about 15% of normal, and there are similar effects on tim protein (TIM). The cycling amplitude of other clock genes is also strongly reduced, in these cases due to increases in trough levels. These data underscore the important contribution of the upstream E-box enhancer region to tim expression and of TIM to clock gene transcriptional repression in fly heads. Surprisingly, tim expression in clock neurons is only modestly affected by the biggest upstream deletion and is similarly affected by a deletion of the intronic E-box region. This distinction between clock neurons and glia is paralleled by a dramatically enhanced accessibility of the intronic enhancer region within clock neurons. This distinctive feature of tim chromatin was revealed by ATAC-seq (assay for transposase-accessible chromatin with sequencing) assays of purified neurons and glia as well as of fly heads. The enhanced cell type-specific accessibility of the intronic enhancer region explains the resilience of clock neuron tim expression and circadian behavior to deletion of the otherwise more prominent upstream tim E-box region.

Visualization of the Association of Dimeric Protein Complexes on Specific Enhancers in the Salivary Gland Nuclei of Drosophila Larva.

Transcription factors (TFs) regulate gene expression by recognizing specific target enhancers in the genome. The DNA-binding and regulatory activity of TFs depend on the presence of additional protein partners, leading to the formation of versatile and dynamic multimeric protein complexes. Visualizing these protein-protein interactions (PPIs) in the nucleus is key for decrypting the molecular cues underlying TF specificity in vivo. Over the last few years, Bimolecular Fluorescence Complementation (BiFC) has been developed in several model systems and applied in the analysis of different types of PPIs. In particular, BiFC has been applied when analyzing PPIs with hundreds of TFs in the nucleus of live Drosophila embryos. However, the visualization of PPIs at the level of specific target enhancers or genomic regions of interest awaits the advent of DNA-labelling methods that can be coupled with BiFC. Here, we present a novel experimental strategy that we have called BiFOR and that is based on the coupling of BiFC with the bacterial ANCHOR DNA-labelling system. We demonstrate that BiFOR enables the precise quantification of the enrichment of specific dimeric protein complexes on target enhancers in Drosophila salivary gland nuclei. Given its versatility and sensitivity, BiFOR could be applied more widely to other tissues during Drosophila development. Our work sets up the experimental basis for future applications of this strategy.

p24 family Tango(1) at the endoplasmic reticulum exit site to organize cargo exit.

The p24 family of proteins have been regarded as cargo receptors for endoplasmic reticulum (ER) to Golgi transport; however, their precise functions have yet to be revealed. In this issue, Pastor-Pareja and colleagues (https://doi.org/10.1083/jcb.202309045) show that the interaction of these proteins with Tango1 is critical for their localization at the ER exit site (ERES) and efficient transport of secretory proteins in Drosophila.

Basal actomyosin pulses expand epithelium coordinating cell flattening and tissue elongation.

Actomyosin networks constrict cell area and junctions to alter cell and tissue shape. However, during cell expansion under mechanical stress, actomyosin networks are strengthened and polarized to relax stress. Thus, cells face a conflicting situation between the enhanced actomyosin contractile properties and the expansion behaviour of the cell or tissue. To address this paradoxical situation, we study late Drosophila oogenesis and reveal an unusual epithelial expansion wave behaviour. Mechanistically, Rac1 and Rho1 integrate basal pulsatile actomyosin networks with ruffles and focal adhesions to increase and then stabilize basal area of epithelial cells allowing their flattening and elongation. This epithelial expansion behaviour bridges cell changes to oocyte growth and extension, while oocyte growth in turn deforms the epithelium to drive cell spreading. Basal pulsatile actomyosin networks exhibit non-contractile mechanics, non-linear structures and F-actin/Myosin-II spatiotemporal signal separation, implicating unreported expanding properties. Biophysical modelling incorporating these expanding properties well simulates epithelial cell expansion waves. Our work thus highlights actomyosin expanding properties as a key mechanism driving tissue morphogenesis.

Formation of recurring transient Ca2+-based intercellular communities during Drosophila hematopoiesis.

Tissue development occurs through a complex interplay between many individual cells. Yet, the fundamental question of how collective tissue behavior emerges from heterogeneous and noisy information processing and transfer at the single-cell level remains unknown. Here, we reveal that tissue scale signaling regulation can arise from local gap-junction mediated cell-cell signaling through the spatiotemporal establishment of an intermediate-scale of transient multicellular communication communities over the course of tissue development. We demonstrated this intermediate scale of emergent signaling using Ca2+ signaling in the intact, ex vivo cultured, live developing Drosophila hematopoietic organ, the lymph gland. Recurrent activation of these transient signaling communities defined self-organized signaling "hotspots" that gradually formed over the course of larva development. These hotspots receive and transmit information to facilitate repetitive interactions with nonhotspot neighbors. Overall, this work bridges the scales between single-cell and emergent group behavior providing key mechanistic insight into how cells establish tissue-scale communication networks.

Cdk8/CDK19 promotes mitochondrial fission through Drp1 phosphorylation and can phenotypically suppress pink1 deficiency in Drosophila.

Cdk8 in Drosophila is the orthologue of vertebrate CDK8 and CDK19. These proteins have been shown to modulate transcriptional control by RNA polymerase II. We found that neuronal loss of Cdk8 severely reduces fly lifespan and causes bang sensitivity. Remarkably, these defects can be rescued by expression of human CDK19, found in the cytoplasm of neurons, suggesting a non-nuclear function of CDK19/Cdk8. Here we show that Cdk8 plays a critical role in the cytoplasm, with its loss causing elongated mitochondria in both muscles and neurons. We find that endogenous GFP-tagged Cdk8 can be found in both the cytoplasm and nucleus. We show that Cdk8 promotes the phosphorylation of Drp1 at S616, a protein required for mitochondrial fission. Interestingly, Pink1, a mitochondrial kinase implicated in Parkinson's disease, also phosphorylates Drp1 at the same residue. Indeed, overexpression of Cdk8 significantly suppresses the phenotypes observed in flies with low levels of Pink1, including elevated levels of ROS, mitochondrial dysmorphology, and behavioral defects. In summary, we propose that Pink1 and Cdk8 perform similar functions to promote Drp1-mediated fission.

Expanding the Drosophila toolkit for dual control of gene expression.

The ability to independently control gene expression in two different tissues in the same animal is emerging as a major need, especially in the context of inter-organ communication studies. This type of study is made possible by technologies combining the GAL4/UAS and a second binary expression system such as the LexA system or QF system. Here, we describe a resource of reagents that facilitate combined use of the GAL4/UAS and a second binary system in various Drosophila tissues. Focusing on genes with well-characterized GAL4 expression patterns, we generated a set of more than 40 LexA-GAD and QF2 insertions by CRISPR knock-in and verified their tissue specificity in larvae. We also built constructs that encode QF2 and LexA-GAD transcription factors in a single vector. Following successful integration of this construct into the fly genome, FLP/FRT recombination is used to isolate fly lines that express only QF2 or LexA-GAD. Finally, using new compatible shRNA vectors, we evaluated both LexA and QF systems for in vivo gene knockdown and are generating a library of such RNAi fly lines as a community resource. Together, these LexA and QF system vectors and fly lines will provide a new set of tools for researchers who need to activate or repress two different genes in an orthogonal manner in the same animal.

In order for researchers to understand how organisms develop and function, they often switch specific genes on or off in certain tissues or at selected times. This can be achieved using genetic tools called binary expression systems. In the fruit fly ­ a popular organism for studying biological processes ­ the most common is the GAL4/UAS system. In this system, a protein called GAL4 is expressed in a specific organ or tissue where it activates a UAS element ­ a genetic sequence that is inserted in front of the gene that is to be switched on. This can also include genes inserted into the fruit fly encoding fluorescent proteins or stretches of DNA coding for factors that can silence specific genes. For example, fruit flies expressing GAL4 protein specifically in nerve cells and a UAS element in front of a gene for a fluorescent protein will display fluorescent nerve cells, which can then be examined using fluorescence microscopy. Studying how organs communicate with one other can require controlled expression of multiple genes at the same time. In fruit flies, other binary expression systems that are analogous to the GAL4/UAS system (known as LexA/LexAop and QF/QUAS) can be used in tandem. For example, to study gut-brain communication, the GAL4/UAS system might be used to switch on the gene for an insulin-like protein in the gut, with one of the other systems controlling the expression of its corresponding receptor in the brain. However, these experiments are currently difficult because, while there are thousands of GAL4/UAS genetic lines, there are only a few LexA/LexAop and QF/QUAS genetic lines. To address this lack of resources, Zirin et al. produced a range of genetically engineered fruit flies containing the LexA/LexAop and QF/QUAS binary expression systems. The flies expressed LexA or QF in each of the major fly organs, including the brain, heart, muscles, and gut. A fluorescent reporter gene linked to the LexAop or QUAS elements, respectively, was then used to test the specificity to single organs and compare the different systems. In some organs the LexA/LexAop system was more reliable than the QF/QUAS system. However, both systems could be successfully combined with genetic elements to switch on a fluorescent reporter gene or switch off a gene of interest in the intended organ. The resources developed by Zirin et al. expand the toolkit for studying fruit fly biology. In future, it will be important to understand the differences between GAL4, LexA and QF systems, and to increase the number of fruit fly lines containing the newer binary expression systems.

Molecular circadian rhythms are robust in marine annelids lacking rhythmic behavior.

The circadian clock controls behavior and metabolism in various organisms. However, the exact timing and strength of rhythmic phenotypes can vary significantly between individuals of the same species. This is highly relevant for rhythmically complex marine environments where organismal rhythmic diversity likely permits the occupation of different microenvironments. When investigating circadian locomotor behavior of Platynereis dumerilii, a model system for marine molecular chronobiology, we found strain-specific, high variability between individual worms. The individual patterns were maintained for several weeks. A diel head transcriptome comparison of behaviorally rhythmic versus arrhythmic wild-type worms showed that 24-h cycling of core circadian clock transcripts is identical between both behavioral phenotypes. While behaviorally arrhythmic worms showed a similar total number of cycling transcripts compared to their behaviorally rhythmic counterparts, the annotation categories of their transcripts, however, differed substantially. Consistent with their locomotor phenotype, behaviorally rhythmic worms exhibit an enrichment of cycling transcripts related to neuronal/behavioral processes. In contrast, behaviorally arrhythmic worms showed significantly increased diel cycling for metabolism- and physiology-related transcripts. The prominent role of the neuropeptide pigment-dispersing factor (PDF) in Drosophila circadian behavior prompted us to test for a possible functional involvement of Platynereis pdf. Differing from its role in Drosophila, loss of pdf impacts overall activity levels but shows only indirect effects on rhythmicity. Our results show that individuals arrhythmic in a given process can show increased rhythmicity in others. Across the Platynereis population, rhythmic phenotypes exist as a continuum, with no distinct "boundaries" between rhythmicity and arrhythmicity. We suggest that such diel rhythm breadth is an important biodiversity resource enabling the species to quickly adapt to heterogeneous or changing marine environments. In times of massive sequencing, our work also emphasizes the importance of time series and functional tests.

CO2 exposure drives a rapid pH response in live adult Drosophila.

CO2 anesthesia is the most common method for immobilizing Drosophila for research purposes. But CO2 exposure has consequences-it can impact fertility, behavior, morphogenesis, and cytoskeletal dynamics. In this respect, Drosophila is an outstanding model for studying the impact of CO2 exposure on tissues. In this study we explored the response of intracellular pH (pHi) to a one-minute CO2 pulse using a genetically encoded, ubiquitously expressed pH sensor, tpHusion, to monitor pHi within a live, intact, whole fly. We compared wild-type flies to flies lacking Imaginal disc growth factors (Idgfs), which are chitinase-like proteins that facilitate developmental processes and the innate immune response. Morphogenetic and cytoskeletal defects in Idgf-null flies are enhanced after CO2 exposure. We found that pHi drops sharply within seconds of the beginning of a CO2 pulse and recovers over several minutes. The initial profile was nearly identical in control and Idgf-null flies but diverged as the pHi returned to normal. This study demonstrates the feasibility of monitoring pH in live adult Drosophila. Studies exploring pH homeostasis are important for understanding human pathologies associated with pH dysregulation.

Experience-dependent glial pruning of synaptic glomeruli during the critical period.

Critical periods are temporally-restricted, early-life windows when sensory experience remodels synaptic connectivity to optimize environmental input. In the Drosophila juvenile brain, critical period experience drives synapse elimination, which is transiently reversible. Within olfactory sensory neuron (OSN) classes synapsing onto single projection neurons extending to brain learning/memory centers, we find glia mediate experience-dependent pruning of OSN synaptic glomeruli downstream of critical period odorant exposure. We find glial projections infiltrate brain neuropil in response to critical period experience, and use Draper (MEGF10) engulfment receptors to prune synaptic glomeruli. Downstream, we find antagonistic Basket (JNK) and Puckered (DUSP) signaling is required for the experience-dependent translocation of activated Basket into glial nuclei. Dependent on this signaling, we find critical period experience drives expression of the F-actin linking signaling scaffold Cheerio (FLNA), which is absolutely essential for the synaptic glomeruli pruning. We find Cheerio mediates experience-dependent regulation of the glial F-actin cytoskeleton for critical period remodeling. These results define a sequential pathway for experience-dependent brain synaptic glomeruli pruning in a strictly-defined critical period; input experience drives neuropil infiltration of glial projections, Draper/MEGF10 receptors activate a Basket/JNK signaling cascade for transcriptional activation, and Cheerio/FLNA induction regulates the glial actin cytoskeleton to mediate targeted synapse phagocytosis.

Knock-in mutagenesis in Drosophila Rdl underscores the critical role of the conserved M3 glycine in mediating the actions of broflanilide and isocycloseram on GABA receptors.

γ-Aminobutyric acid receptors (GABARs) are crucial targets for pest control chemicals, including meta-diamide and isoxazoline insecticides, which act as negative allosteric modulators of insect GABARs. Previous cell-based assays have indicated that amino acid residues in the transmembrane cavity between adjacent subunits of Drosophila RDL GABAR (i.e., Ile276, Leu280, and Gly335) are involved in mediating the action of meta-diamides. In this study, to confirm this result at the organismal level, we employed CRISPR/Cas9-mediated genome editing, generated six transgenic Drosophila strains carrying substitutions in these amino acid residues, and investigated their sensitivity to broflanilide and isocycloseram. Flies homozygous for the I276F mutation did not exhibit any change in sensitivity to the tested insecticides compared to the control flies. Conversely, I276C homozygosity was lethal, and heterozygous flies exhibited ∼2-fold lower sensitivity to broflanilide than the control flies. Flies homozygous for the L280C mutation survived into adulthood but exhibited infertility. Both heterozygous and homozygous L280C flies exhibited ∼3- and âˆ¼20-fold lower sensitivities to broflanilide and isocycloseram, respectively, than the control flies. The reduction in sensitivity to isocycloseram in L280C flies diminished to ∼3-fold when treated with piperonyl butoxide. Flies homozygous for the G335A mutation reached the adult stage. However, they were sterile, had small bodies, and exhibited reduced locomotion, indicating the critical role of Gly335 in RDL function. These flies exhibited markedly increased tolerance to topically applied broflanilide and isocycloseram, demonstrating that the conserved Gly335 is the target of the insecticidal actions of broflanilide and isocycloseram. Considering the significant fitness costs, the Gly335 mutation may not pose a serious risk for the development of resistance in field populations of insect pests. However, more careful studies using insect pests are needed to investigate whether our perspective applies to resistance development under field conditions.

Balancing competing effects of tissue growth and cytoskeletal regulation during Drosophila wing disc development.

How a developing organ robustly coordinates the cellular mechanics and growth to reach a final size and shape remains poorly understood. Through iterations between experiments and model simulations that include a mechanistic description of interkinetic nuclear migration, we show that the local curvature, height, and nuclear positioning of cells in the Drosophila wing imaginal disc are defined by the concurrent patterning of actomyosin contractility, cell-ECM adhesion, ECM stiffness, and interfacial membrane tension. We show that increasing cell proliferation via different growth-promoting pathways results in two distinct phenotypes. Triggering proliferation through insulin signaling increases basal curvature, but an increase in growth through Dpp signaling and Myc causes tissue flattening. These distinct phenotypic outcomes arise from differences in how each growth pathway regulates the cellular cytoskeleton, including contractility and cell-ECM adhesion. The coupled regulation of proliferation and cytoskeletal regulators is a general strategy to meet the multiple context-dependent criteria defining tissue morphogenesis.

Lethal Giant Disc is a target of Cdk1 and regulates ESCRT-III localization during germline stem cell abscission.

Abscission is the final step of cytokinesis that allows the physical separation of sister cells through the scission of the cellular membrane. This deformation is driven by ESCRT-III proteins, which can bind membranes and form dynamic helices. A crucial step in abscission is the recruitment of ESCRT-III proteins at the right time and place. Alix is one of the best characterized proteins that recruits ESCRT-III proteins from yeast to mammals. However, recent studies in vivo have revealed that pathways acting independently or redundantly with Alix are also required at abscission sites in different cellular contexts. Here, we show that Lgd acts redundantly with Alix to properly localize ESCRT-III to the abscission site in germline stem cells (GSCs) during Drosophila oogenesis. We further demonstrate that Lgd is phosphorylated at multiple sites by the CycB/Cdk1 kinase. We found that these phosphorylation events potentiate the activity of Shrub, a Drosophila ESCRT-III, during abscission of GSCs. Our study reveals that redundancy between Lgd and Alix, and coordination with the cell cycle kinase Cdk1, confers robust and timely abscission of Drosophila germline stem cells.

Identification of immunity-related genes distinctly regulated by Manduca sexta Spӓtzle-1/2 and Escherichia coli peptidoglycan.

The immune system of Manduca sexta has been well studied to understand molecular mechanisms of insect antimicrobial responses. While evidence supports the existence of major immune signaling pathways in this species, it is unclear how induced production of defense proteins is specifically regulated by the Toll and Imd pathways. Our previous studies suggested that diaminopimelic acid-type peptidoglycans (DAP-PG) from Gram-negative and some Gram-positive bacteria, more than Lys-type peptidoglycans (Lys-PG) from other Gram-positive bacteria, triggers both pathways through membrane-bound receptors orthologous to Drosophila Toll and PGRP-LC. In this study, we produced M. sexta proSpätzle-1 and proSpätzle-2 in Sf9 cells, identified their processing enzymes, and used prophenoloxidase activating protease-3 to activate the cytokine precursors. After Spätzle-1 and -2 were isolated from the reaction mixtures, we separately injected the purified cytokines into larval hemocoel to induce gene transcription in fat body through the Toll pathway solely. On the other hand, we treated a M. sexta cell line with E. coli DAP-PG to only induce the Imd pathway and target gene expression. RNA-Seq analysis of the fat body and cultured cells collected at 0, 6, and 24 h after treatment indicated that expression of diapausin-4, -10, -12, -13, cecropin-2, -4, -5, attacin-5, -11, and lebocin D is up-regulated predominantly via Toll signaling, whereas transcription of cecropin-6, gloverin, lysozyme-1, and gallerimycin-2 is mostly induced by DAP-PG via Imd signaling. Other antimicrobial peptides are expressed in response to both pathways. Transcripts of most Toll-specific genes (e.g., lebocin D) peaked at 6 h, contrasting the gradual increase and plateauing of drosomycin mRNA level at 24-48 h in Drosophila. We also used T (oll)-I (md) ratios to estimate relative contributions of the two pathways to transcriptional regulation of other components of the immune system. The differences in pathway specificity and time course of transcriptional regulation call for further investigations in M. sexta and other insects.

Determination of the larval precursor configuration of the Drosophila adult hindgut by G-TRACE analysis.

The Drosophila hindgut is a classical model to study organogenesis. The adult hindgut originates from the precursor cells in the larval hindgut. However, the territory of these cells has still not been well determined. A ring of wingless (wg)-expressing cells lies at the anterior zone of both the larval and adult hindgut. The larval Wg ring was thought as a portion of precursor of the adult hindgut. By applying a cell lineage tracing tool (G-TRACE), we demonstrate that larval wg-expressing cells have no cell lineage contribution to the adult hindgut. Additionally, adult Wg ring cells do not divide and move posteriorly to replenish the hindgut tissue. Instead, we determine that the precursors of the adult pylorus and ileum are situated in the cubitus interruptus (ci)-expressing cells in the anterior zone, and deduce that the precursor stem cells of the adult rectum locate in the trunk region of the larval pylorus including hedgehog (hh)-expressing cells. Together, this research advances our understanding of cell lineage origins and the development of the Drosophila hindgut.

Dual treatment with kynurenine pathway inhibitors and NAD+ precursors synergistically extends life span in Drosophila.

Tryptophan catabolism is highly conserved and generates important bioactive metabolites, including kynurenines, and in some animals, NAD+. Aging and inflammation are associated with increased levels of kynurenine pathway (KP) metabolites and depleted NAD+, factors which are implicated as contributors to frailty and morbidity. Contrastingly, KP suppression and NAD+ supplementation are associated with increased life span in some animals. Here, we used DGRP_229 Drosophila to elucidate the effects of KP elevation, KP suppression, and NAD+ supplementation on physical performance and survivorship. Flies were chronically fed kynurenines, KP inhibitors, NAD+ precursors, or a combination of KP inhibitors with NAD+ precursors. Flies with elevated kynurenines had reduced climbing speed, endurance, and life span. Treatment with a combination of KP inhibitors and NAD+ precursors preserved physical function and synergistically increased maximum life span. We conclude that KP flux can regulate health span and life span in Drosophila and that targeting KP and NAD+ metabolism can synergistically increase life span.

Spatio-temporal distribution and genetic background of elastic proteins inside the chitin/chitosan matrix of insects including their functional significance for locomotion.

In insects, cuticle proteins interact with chitin and chitosan of the exoskeleton forming crystalline, amorphic or composite material structures. The biochemical and mechanical composition of the structure defines the cuticle's physical properties and thus how the insect cuticle behaves under mechanical stress. The tissue-specific ratio between chitin and chitosan and its pattern of deacetylation are recognized and interpreted by cuticle proteins depending on their local position in the body. Despite previous research, the assembly of the cuticle composites in time and space including its functional impact is widely unexplored. This review is devoted to the genetics underlying the temporal and spatial distribution of elastic proteins and the potential function of elastic proteins in insects with a focus on Resilin in the fruit fly Drosophila. The potential impact and function of localized patches of elastic proteins is discussed for movements in leg joints, locomotion and damage resistance of the cuticle. We conclude that an interdisciplinary research approach serves as an integral example for the molecular mechanisms of generation and interpretation of the chitin/chitosan matrix, not only in Drosophila but also in other arthropod species, and might help to synthesize artificial material composites.

Role of RNA binding proteins of the Drosophila behavior and human splicing (DBHS) family in health and cancer.

RNA-binding proteins (RBPs) play crucial roles in the functions and homoeostasis of various tissues by regulating multiple events of RNA processing including RNA splicing, intracellular RNA transport, and mRNA translation. The Drosophila behavior and human splicing (DBHS) family proteins including PSF/SFPQ, NONO, and PSPC1 are ubiquitously expressed RBPs that contribute to the physiology of several tissues. In mammals, DBHS proteins have been reported to contribute to neurological diseases and play crucial roles in cancers, such as prostate, breast, and liver cancers, by regulating cancer-specific gene expression. Notably, in recent years, multiple small molecules targeting DBHS family proteins have been developed for application as cancer therapeutics. This review provides a recent overview of the functions of DBHS family in physiology and pathophysiology, and discusses the application of DBHS family proteins as promising diagnostic and therapeutic targets for cancers.

The Drosophila neuroblast polarity cycle at a glance.

Drosophila neural stem cells, or neuroblasts, rapidly proliferate during embryonic and larval development to populate the central nervous system. Neuroblasts divide asymmetrically to create cellular diversity, with each division producing one sibling cell that retains the neuroblast fate and another that differentiates into glia or neurons. This asymmetric outcome is mediated by the transient polarization of numerous factors to the cell cortex during mitosis. The powerful genetics and outstanding imaging tractability of the neuroblast make it an excellent model system for studying the mechanisms of cell polarity. This Cell Science at a Glance article and the accompanying poster explore the phases of the neuroblast polarity cycle and the regulatory circuits that control them. We discuss the key features of the cycle - the targeted recruitment of proteins to specific regions of the plasma membrane and multiple phases of highly dynamic actomyosin-dependent cortical flows that pattern both protein distribution and membrane structure.

Communication between the stem cell niche and an adjacent differentiation niche through miRNA and EGFR signaling orchestrates exit from the stem cell state in the Drosophila ovary.

The signaling environment, or niche, often governs the initial difference in behavior of an adult stem cell and a derivative that initiates a path towards differentiation. The transition between an instructive stem cell niche and differentiation niche must generally have single-cell resolution, suggesting that multiple mechanisms might be necessary to sharpen the transition. Here, we examined the Drosophila ovary and found that Cap cells, which are key constituents of the germline stem cell (GSC) niche, express a conserved microRNA (miR-124). Surprisingly, loss of miR-124 activity in Cap cells leads to a defect in differentiation of GSC derivatives. We present evidence that the direct functional target of miR-124 in Cap cells is the epidermal growth factor receptor (EGFR) and that failure to limit EGFR expression leads to the ectopic expression of a key anti-differentiation BMP signal in neighboring somatic escort cells (ECs), which constitute a differentiation niche. We further found that Notch signaling connects EFGR activity in Cap cells to BMP expression in ECs. We deduce that the stem cell niche communicates with the differentiation niche through a mechanism that begins with the selective expression of a specific microRNA and culminates in the suppression of the major anti-differentiation signal in neighboring cells, with the functionally important overall role of sharpening the spatial distinction between self-renewal and differentiation environments.