Ruifen Weng

Systematic Study of Drosophila MicroRNA Functions Using a Collection of Targeted Knockout Mutations

MicroRNAs 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.

Drosophila miR-124 regulates neuroblast proliferation through its target anachronism

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.

Overlapping functions of microRNAs in control of apoptosis during Drosophila embryogenesis

Regulation of apoptosis is crucial for tissue homeostasis under normal development and environmental stress. In Drosophila, cell death occurs in different developmental processes including embryogenesis. Here, we report that two members of the miR-2 seed family of microRNAs, miR-6 and miR-11, function together to limit the level of apoptosis during Drosophila embryonic development. Mutants lacking both miR-6 and miR-11 show embryonic lethality and defects in the central nervous system (CNS). We provide evidence that miR-6/11 functions through regulation of the proapoptotic genes, reaper (rpr), head involution defective (hid), grim and sickle (skl). Upregulation of these proapoptotic genes is responsible for the elevated apoptosis and the CNS defects in the mutants. These findings demonstrate that the activity of the proapoptotic genes is kept in check by miR-6/11 to ensure normal development.

Recombinase-Mediated Cassette Exchange Provides a Versatile Platform for Gene Targeting: Knockout of miR-31b

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.

The Oscillating miRNA 959-964 Cluster Impacts Drosophila Feeding Time and Other Circadian Outputs

We 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.

Coordination of insulin and Notch pathway activities by microRNA miR-305 mediates adaptive homeostasis in the intestinal stem cells of the Drosophila gut

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.

miR-124 controls male reproductive success in Drosophila

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

Crosstalk between Epithelial and Mesenchymal Tissues in Tumorigenesis and Imaginal Disc Development

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.

Maternal loss of miRNAs leads to increased variance in primordial germ cell numbers in Drosophila melanogaster.

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.

miR-989 is required for border cell migration in the Drosophila ovary.

microRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by destabilizing target transcripts and/or inhibiting their translation. miRNAs are thought to have roles in buffering gene expression to confer robustness. miRNAs have been shown to play important roles during tissue development to control cell proliferation, differentiation and morphogenesis. Many miRNAs are expressed in the germ line of Drosophila, and functions have been reported for a few miRNAs in maintenance of stem cell proliferation during oogenesis. Here, we analyse the function of Drosophila miR-989 in oogenesis. miR-989 is abundant in ovaries. Mutants lacking miR-989 did not display gross abnormalities affecting egg chamber formation or maturation. However, the migration of the border cell cluster was severely delayed in miR-989 mutant egg chambers. We demonstrate that miR-989 function is required in the somatic cells in the egg chamber, not in germ line cells for border cell migration. Loss of miR-989 from a fraction of the border cell cluster was sufficient to impair cluster migration as a whole, suggesting a role in border cells. Gene ontology analysis reveals that many predicted miR-989 target mRNAs are implicated in regulating cell migration, cell projection morphogenesis, cell adhesion as well as receptor tyrosine kinase and ecdysone signalling, consistent with an important regulatory role for miR-989 in border cell migration.