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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., Summary: The Drosophila miRNA bantam regulates the expression of Brat and Prospero – known inhibitors of brain neuroblast proliferation – to modulate growth of the central brain.

SummaryMicroRNAs are abundant in animal genomes, yet little is known about their functions in vivo. Here, we report the production of 80 new Drosophila miRNA mutants by targeted homologous recombination. These mutants remove 104 miRNAs. Together with 15 previously reported mutants, this collection includes 95 mutants deleting 130 miRNAs. Collectively, these genes produce over 99% of all Drosophila miRNAs, measured by miRNA sequence reads. We present a survey of developmental and adult miRNA phenotypes. Over 80% of the mutants showed at least one phenotype using a p < 0.01 significance threshold. We observed a significant correlation between miRNA abundance and phenotypes related to survival and lifespan, but not to most other phenotypes. miRNA cluster mutants were no more likely than single miRNA mutants to produce significant phenotypes. This mutant collection will provide a resource for future analysis of the biological roles of Drosophila miRNAs.

MicroRNAs (miRNAs) are posttranscriptional regulators of gene expression that may act as buffering agents to stabilize gene-regulatory networks. Here, we identify two miRNAs that are maternally required for normal embryonic primordial germ cell development in Drosophila melanogaster. Embryos derived from miR-969 and miR-9c mutant mothers had, on average, reduced germ cell numbers. Intriguingly, this reduction correlated with an increase in the variance of this quantitative phenotypic trait. Analysis of an independent set of maternal mutant genotypes suggests that reduction of germ cell number need not lead to increased variance. Our observations are consistent with the hypothesis that miR-969 and miR-9c contribute to stabilizing the processes that control germ number, supporting phenotypic robustness.

SummaryWe sequenced Drosophila head RNA to identify a small set of miRNAs that undergo robust circadian cycling. We concentrated on a cluster of six miRNAs, mir-959-964, all of which peak at about ZT12 or lights off. The cluster pri-miRNA is transcribed under bona fide circadian transcriptional control, and all six mature miRNAs have short half-lives, a requirement for cycling. A viable Gal4 knockin strain localizes prominent cluster miRNA expression to the adult head fat body. Analysis of cluster knockout and overexpression strains indicates that innate immunity, metabolism, and feeding behavior are under cluster miRNA regulation. Manipulation of food intake also affects the levels and timing of cluster miRNA transcription with no more than minor effects on the core circadian oscillator. These observations indicate a feedback circuit between feeding time and cluster miRNA expression function as well as a surprising role of posttranscriptional regulation in the circadian control of these phenotypes.

A series of vectors has been designed to enhance the versatility of targeted homologous recombination. Recombinase-mediated cassette exchange permits sequential targeting at any locus and improves flexibility in making user-defined mutations. Application of RMCE to delete an intronic microRNA gene is described.

Homeostasis of the intestine is maintained by dynamic regulation of a pool of intestinal stem cells. The balance between stem cell self-renewal and differentiation is regulated by the Notch and insulin signaling pathways. Dependence on the insulin pathway places the stem cell pool under nutritional control, allowing gut homeostasis to adapt to environmental conditions. Here we present evidence that miR-305 is required for adaptive homeostasis of the gut. miR-305 regulates the Notch and insulin pathways in the intestinal stem cells. Notably, miR-305 expression in the stem cells is itself under nutritional control via the insulin pathway. This link places regulation of Notch pathway activity under nutritional control. These findings provide a mechanism through which the insulin pathway controls the balance between stem cell self-renewal and differentiation that is required for adaptive homeostasis in the gut in response to changing environmental conditions.

Summary Background Cancers develop in a complex mutational landscape. Interaction of genetically abnormal cancer cells with normal stromal cells can modify the local microenvironment to promote disease progression for some tumor types. Genetic models of tumorigenesis provide the opportunity to explore how combinations of cancer driver mutations confer distinct properties on tumors. Previous Drosophila models of EGFR-driven cancer have focused on epithelial neoplasia. Results Here, we report a Drosophila genetic model of EGFR-driven tumorigenesis in which the neoplastic transformation depends on interaction between epithelial and mesenchymal cells. We provide evidence that the secreted proteoglycan Perlecan can act as a context-dependent oncogene cooperating with EGFR to promote tumorigenesis. Coexpression of Perlecan in the EGFR-expressing epithelial cells potentiates endogenous Wg/Wnt and Dpp/BMP signals from the epithelial cells to support expansion of a mesenchymal compartment. Wg activity is required in the epithelial compartment, whereas Dpp activity is required in the mesenchymal compartment. This genetically normal mesenchymal compartment is required to support growth and neoplastic transformation of the genetically modified epithelial population. Conclusions We report a genetic model of tumor formation that depends on crosstalk between a genetically modified epithelial cell population and normal host mesenchymal cells. Tumorigenesis in this model co-opts a regulatory mechanism that is normally involved in controlling growth of the imaginal disc during development.

Pushpa Verma's name was inadvertently omitted in the list of author list for the article. The authors would like to revise the author list to read as below: Jan-Michael Kugler, Pushpa Verma, Ya-Wen Chen, Ruifen Weng, Stephen M.Cohen Pushpa Verma 's affiliation is Institute of Molecular and Cell Biology, Singapore.

Many aspects of social behavior are controlled by sex-specific pheromones. Gender-appropriate production of the sexually dimorphic transcription factors doublesex and fruitless controls sexual differentiation and sexual behavior. miR-124 mutant males exhibited increased male–male courtship and reduced reproductive success with females. Females showed a strong preference for wild-type males over miR-124 mutant males when given a choice of mates. These effects were traced to aberrant pheromone production. We identified the sex-specific splicing factor transformer as a functionally significant target of miR-124 in this context, suggesting a role for miR-124 in the control of male sexual differentiation and behavior, by limiting inappropriate expression of the female form of transformer. miR-124 is required to ensure fidelity of gender-appropriate pheromone production in males. Use of a microRNA provides a secondary means of controlling the cascade of sex-specific splicing events that controls sexual differentiation in Drosophila. DOI: http://dx.doi.org/10.7554/eLife.00640.001, eLife digest Like many animals, the fruit fly Drosophila uses pheromones to influence sexual behaviour, with males and females producing different versions of these chemicals. One of the pheromones produced by male flies, for example, is a chemical called 11-cis-vaccenyl-acetate (cVA), which is an aphrodisiac for female flies and an anti-aphrodisiac for males. The production of the correct pheromones in each sex is genetically controlled using a process called splicing that allows a single gene to be expressed as two or more different proteins. A variety of proteins called splicing factors ensures that splicing results in the production of the correct pheromones for each sex. Sometimes, however, the process by which sex genes are expressed as proteins can be ‘leaky’, which results in the wrong proteins being produced for one or both sexes. Small RNA molecules called microRNAs act in some genetic pathways to limit the leaky expression of genes, and a microRNA called miR-124 carries out this function in the developing brain Drosophila. Now, Weng et al. show that miR-124 also helps to regulate sex-specific splicing and thereby to control pheromone production and sexual behaviour. Mutant male flies lacking miR-124 were less successful than wild-type males at mating with female flies, and were almost always rejected if a female fly was given a choice between a mutant male and a wild-type male. Moreover, both wild-type and mutant male flies were more likely to initiate courtship behaviour towards another male if it lacked miR-124 than if it did not. The mutant male flies produced less cVA than wild-type males, but more of other pheromones called pentacosenes, which is consistent with the observed behaviour because cVA attracts females and repels males, whereas pentacosenes act as aphrodisiacs for male flies in large amounts. Weng et al. showed that these changes in the production of pheromones were caused by an increased expression of the female version of a splicing factor called transformer in the mutant males, but further work is needed to understand this process in detail. DOI: http://dx.doi.org/10.7554/eLife.00640.002

MicroRNAs (miRNAs) have been implicated as regulators of central nervous system (CNS) development and function. miR-124 is an evolutionarily ancient, CNS-specific miRNA. On the basis of the evolutionary conservation of its expression in the CNS, miR-124 is expected to have an ancient conserved function. Intriguingly, investigation of miR-124 function using antisense-mediated miRNA depletion has produced divergent and in some cases contradictory findings in a variety of model systems. Here we investigated miR-124 function using a targeted knockout mutant and present evidence for a role during central brain neurogenesis in Drosophila melanogaster. miR-124 activity in the larval neuroblast lineage is required to support normal levels of neuronal progenitor proliferation. We identify anachronism (ana), which encodes a secreted inhibitor of neuroblast proliferation, as a functionally important target of miR-124 acting in the neuroblast lineage. ana has previously been thought to be glial specific in its expression and to act from the cortex glia to control the exit of neuroblasts from quiescence into the proliferative phase that generates the neurons of the adult CNS during larval development. We provide evidence that ana is expressed in miR-124-expressing neuroblast lineages and that ana activity must be limited by the action of miR-124 during neuronal progenitor proliferation. We discuss the possibility that the apparent divergence of function of miR-124 in different model systems might reflect functional divergence through target site evolution.

MicroRNAs (miRNAs) are noncoding RNA molecules that have come to attract considerable interest for their roles in animal and plant development and disease. One means to study miRNA function in animal development is to create mutations. Use of gene-targeting strategies based on ends-out homologous recombination is a useful approach to produce mutations of desired structure, and is gaining popularity for producing miRNA knockouts. Here we present a detailed protocol for miRNA gene targeting and for their subsequent molecular characterization as well as confirmation by rescue. The descriptions of a series of modified vectors designed to facilitate the analysis of miRNA function are included, and a method to manipulate the mutant genome using recombinase-mediated cassette exchange.

MicroRNAs (miRNAs) are noncoding RNA molecules that have come to attract considerable interest for their roles in animal and plant development and disease. One means to study miRNA function in animal development is to create mutations. Use of gene-targeting strategies based on ends-out homologous recombination is a useful approach to produce mutations of desired structure, and is gaining popularity for producing miRNA knockouts. Here we present a detailed protocol for miRNA gene targeting and for their subsequent molecular characterization as well as confirmation by rescue. The descriptions of a series of modified vectors designed to facilitate the analysis of miRNA function are included, and a method to manipulate the mutant genome using recombinase-mediated cassette exchange.