MicroRNA mediated regulation of the onset of enteroblast differentiation in the Drosophila adult intestine.

Somatic adult stem cell lineages in high-turnover tissues are under tight gene regulatory control. Like its mammalian counterpart, the Drosophila intestine precisely adjusts the rate of stem cell division with the onset of differentiation based on physiological demand. Although Notch signaling is indispensable for these decisions, the regulation of Notch activity that drives the differentiation of stem cell progenies into functional, mature cells is not well understood.

Resources and Methods for the Analysis of MicroRNA Function in Drosophila.

Since the widespread discovery of microRNAs (miRNAs) 20 years ago, the Drosophila melanogaster model system has made important contributions to understanding the biology of this class of noncoding RNAs. These contributions are based on the amenability of this model system not only for biochemical analysis but molecular, genetic, and cell biological analyses as well. Nevertheless, while the Drosophila genome is now known to encode 258 miRNA precursors, the function of only a small minority of these have been well characterized.

Molecular Dissection of a Conserved Cluster of miRNAs Identifies Critical Structural Determinants That Mediate Differential Processing.

Differential processing is a hallmark of clustered microRNAs (miRNAs) and the role of position and order of miRNAs in a cluster together with the contribution of stem-base and terminal loops has not been explored extensively within the context of a polycistronic transcript. To elucidate the structural attributes of a polycistronic transcript that contribute towards the differences in efficiencies of processing of the co-transcribed miRNAs, we constructed a series of chimeric variants of that encodes three evolutionary conserved and differentially expressed miRNAs (, and ) and examined the expression and biological activity of the encoded miRNAs. The kinetic effects of Drosha and Dicer processing on the chimeric precursors were examined by processing assays.

The RNA-binding protein Swm is critical for Drosophila melanogaster intestinal progenitor cell maintenance.

The regulation of stem cell survival, self-renewal, and differentiation is critical for the maintenance of tissue homeostasis. Although the involvement of signaling pathways and transcriptional control mechanisms in stem cell regulation have been extensively investigated, the role of post-transcriptional control is still poorly understood. Here, we show that the nuclear activity of the RNA-binding protein Second Mitotic Wave Missing is critical for Drosophila melanogaster intestinal stem cells and their daughter cells, enteroblasts, to maintain their progenitor cell properties and functions.

Coordinated repression of pro-differentiation genes via P-bodies and transcription maintains Drosophila intestinal stem cell identity.

The role of processing bodies (P-bodies), key sites of post-transcriptional control, in adult stem cells remains poorly understood. Here, we report that adult Drosophila intestinal stem cells, but not surrounding differentiated cells such as absorptive enterocytes (ECs), harbor P-bodies that contain Drosophila orthologs of mammalian P-body components DDX6, EDC3, EDC4, and LSM14A/B. A targeted RNAi screen in intestinal progenitor cells identified 39 previously known and 64 novel P-body regulators, including Patr-1, a gene necessary for P-body assembly.

pathway regulates dietary restriction-dependent enhancement of lifespan in .

Dietary restriction (DR) extends healthy lifespan in diverse species. Age and nutrient-related changes in the abundance of microRNAs (miRNAs) and their processing factors have been linked to organismal longevity. However, the mechanisms by which they modulate lifespan and the tissue-specific role of miRNA-mediated networks in DR-dependent enhancement of lifespan remains largely unexplored.

A stress-responsive miRNA regulates BMP signaling to maintain tissue homeostasis.

Adult organisms must sense and adapt to environmental fluctuations. In high-turnover tissues such as the intestine, these adaptive responses require rapid changes in gene expression that, in turn, likely involve posttranscriptional gene control. However, intestinal-tissue-specific microRNA (miRNA)-mediated regulatory pathways remain unexplored.

I-KCKT allows dissection-free RNA profiling of adult intestinal progenitor cells.

The adult intestinal epithelium is a model system for stem cell biology, but its utility is limited by current biochemical methods that lack cell type resolution. Here, we describe a new proximity-based profiling method that relies upon a GAL4 driver, termed (), that is exclusively expressed in intestinal progenitor cells. This method uses UV crosslinked whole animal frozen powder as its starting material to immunoprecipitate the RNA cargoes of transgenic epitope-tagged RNA binding proteins driven by When applied to the general mRNA-binder, poly(A)-binding protein, the RNA profile obtained by this method identifies 98.

Identification of Split-GAL4 Drivers and Enhancers That Allow Regional Cell Type Manipulations of the Intestine.

The adult midgut is a model epithelial tissue composed of a few major cell types with distinct regional identities. One of the limitations to its analysis is the lack of tools to manipulate gene expression based on these regional identities. To overcome this obstacle, we applied the intersectional split-GAL4 system to the adult midgut and report 653 driver combinations that label cells by region and cell type.

Identification and Characterization of Breakpoints and Mutations on Balancer Chromosomes.

Balancers are rearranged chromosomes used in to maintain deleterious mutations in stable populations, preserve sets of linked genetic elements and construct complex experimental stocks. Here, we assess the phenotypes associated with breakpoint-induced mutations on commonly used third chromosome balancers and show remarkably few deleterious effects. We demonstrate that a breakpoint in causes loss of radiation-induced apoptosis and a breakpoint in causes loss of fucosylation in nervous and intestinal tissue-the latter study providing new markers for intestinal cell identity and challenging previous conclusions about the regulation of fucosylation.

The Microbiome Sets the Stage for Cholera.

Interactions between the eukaryotic host, microbiome members, and invading pathogens help to shape disease outcomes. Using the Drosophila model, Fast et al. identified that Vibrio cholerae acts to inhibit epithelial renewal through complex interactions between the type VI secretion system of V.

Canonical nucleators are dispensable for stress granule assembly in intestinal progenitors.

Stressed cells downregulate translation initiation and assemble membrane-less foci termed stress granules (SGs). Although SGs have been extensively characterized in cultured cells, the existence of such structures in stressed adult stem cell pools remains poorly characterized. Here, we report that the orthologs of the mammalian SG components AGO1, ATX2, CAPRIN, eIF4E, FMRP, G3BP, LIN-28, PABP and TIAR are enriched in adult fly intestinal progenitor cells, where they accumulate in small cytoplasmic messenger ribonucleoprotein complexes (mRNPs).

MicroRNAs Regulate Broad-Z3, Which Together with Chinmo Maintains Adult Lineage Neurons in an Immature State.

During metamorphosis, arrested immature neurons born during larval development differentiate into their functional adult form. This differentiation coincides with the downregulation of two zinc-finger transcription factors, Chronologically Inappropriate Morphogenesis (Chinmo) and the Z3 isoform of Broad (Br-Z3). Here, we show that is regulated by two microRNAs, and , that are activated at the larval-to-pupal transition and are known to also regulate The 3'UTR contains functional binding sites for both and that confers sensitivity to both of these microRNAs, as determined by deletion analysis in reporter assays.

Lactate dehydrogenase and glycerol-3-phosphate dehydrogenase cooperatively regulate growth and carbohydrate metabolism during larval development.

The dramatic growth that occurs during larval development requires rapid conversion of nutrients into biomass. Many larval tissues respond to these biosynthetic demands by increasing carbohydrate metabolism and lactate dehydrogenase (LDH) activity. The resulting metabolic program is ideally suited for synthesis of macromolecules and mimics the manner by which cancer cells rely on aerobic glycolysis.

Opposing Post-transcriptional Control of InR by FMRP and LIN-28 Adjusts Stem Cell-Based Tissue Growth.

Although the intrinsic mechanisms that control whether stem cells divide symmetrically or asymmetrically underlie tissue growth and homeostasis, they remain poorly defined. We report that the RNA-binding protein fragile X mental retardation protein (FMRP) limits the symmetric division, and resulting expansion, of the stem cell population during adaptive intestinal growth in Drosophila. The elevated insulin sensitivity that FMRP-deficient progenitor cells display contributes to their accelerated expansion, which is suppressed by the depletion of insulin-signaling components.

Analysis of MicroRNA Function in Drosophila.

MicroRNAs are short noncoding, ~22-nucleotide RNAs that regulate protein abundance. The growth in our understanding of this class of RNAs has been rapid since their discovery just over a decade ago. We now appreciate that miRNAs are deeply embedded within the genetic networks that control basic features of metazoan cells including their identity, metabolism, and reproduction.

A let-7-to-miR-125 MicroRNA Switch Regulates Neuronal Integrity and Lifespan in Drosophila.

Messenger RNAs (mRNAs) often contain binding sites for multiple, different microRNAs (miRNAs). However, the biological significance of this feature is unclear, since such co-targeting miRNAs could function coordinately, independently, or redundantly with one another. Here, we show that two co-transcribed Drosophila miRNAs, let-7 and miR-125, non-redundantly regulate a common target, the transcription factor Chronologically Inappropriate Morphogenesis (Chinmo).

Neural stem cell-encoded temporal patterning delineates an early window of malignant susceptibility in Drosophila.

Pediatric neural tumors are often initiated during early development and can undergo very rapid transformation. However, the molecular basis of this early malignant susceptibility remains unknown. During Drosophila development, neural stem cells (NSCs) divide asymmetrically and generate intermediate progenitors that rapidly differentiate in neurons.

Starving for more: Nutrient sensing by LIN-28 in adult intestinal progenitor cells.

In this Extra View, we extend our recent work on the protein LIN-28 and its role in adult stem cell divisions. LIN-28 is an mRNA- and microRNA-binding protein that is conserved from worms to humans. When expressed ectopically, it promotes the reprogramming of differentiated vertebrate cells into pluripotent stem cells as well as the regeneration of vertebrate tissues after injury.

Lin-28 promotes symmetric stem cell division and drives adaptive growth in the adult Drosophila intestine.

Stem cells switch between asymmetric and symmetric division to expand in number as tissues grow during development and in response to environmental changes. The stem cell intrinsic proteins controlling this switch are largely unknown, but one candidate is the Lin-28 pluripotency factor. A conserved RNA-binding protein that is downregulated in most animals as they develop from embryos to adults, Lin-28 persists in populations of adult stem cells.

ADAR mediates differential expression of polycistronic microRNAs.

Adenosine deaminases acting on RNAs (ADARs) convert adenosine residues to inosines in primary microRNA (pri-miRNA) transcripts to alter the structural conformation of these precursors and the subsequent functions of the encoded microRNAs (miRNAs). Here we show that RNA editing by Drosophila ADAR modulates the expression of three co-transcribed miRNAs encoded by the evolutionarily conserved let-7-Complex (let-7-C) locus. For example, a single A-to-I change at the -6 residue of pri-miR-100, the first miRNA in this let-7-C polycistronic transcript, leads to enhanced miRNA processing by Drosha and consequently enhanced functional miR-100 both in vitro as well as in vivo.

Drosha-independent DGCR8/Pasha pathway regulates neuronal morphogenesis.

Cleavage of microRNAs and mRNAs by Drosha and its cofactor Pasha/DGCR8 is required for animal development, but whether these proteins also have independent roles in development has been unclear. Known phenotypes associated with loss of either one of these two proteins are very similar and consistent with their joint function, even though both cofactors are involved with additional distinct RNA biogenesis pathways. Here, we report clear phenotypic differences between drosha and pasha/dgcr8 null alleles in two postembryonic lineages in the Drosophila brain: elimination of pasha/dgcr8 leads to defects that are not shared by drosha null mutations in the morphology of gamma neurons in the mushroom body lineage, as well as many neurons in the anterodorsal projection neuron lineage.

MicroRNAs as components of systemic signaling pathways in Drosophila melanogaster.

MicroRNAs (miRNAs) ensure progression through development by synchronizing cell fate transitions in response to environmental cues. These cues are mediated at least in part by steroid hormones. Emerging evidence indicates that miRNAs are also components of additional systemic signaling pathways, including insulin, stress, immune, and circadian pathways.

Let-7-complex microRNAs regulate the temporal identity of Drosophila mushroom body neurons via chinmo.

Many neural lineages display a temporal pattern, but the mechanisms controlling the ordered production of neuronal subtypes remain unclear. Here, we show that Drosophila let-7 and miR-125, cotranscribed from the let-7-Complex (let-7-C) locus, regulate the transcription factor chinmo to control temporal cell fate in the mushroom body (MB) lineage. We find that let-7-C is activated in postmitotic neurons born during the larval-to-pupal transition, when transitions among three MB subtypes occur.

Small temporal RNAs in animal development.

The lin-4/miR-125 and let-7 microRNAs are at the heart of the heterochronic pathway, which controls temporal cell fate determination during Caenorhabditis elegans development. These small temporal RNAs are clustered along with a third microRNA, miR-100, in the genomes of most animals. Their conserved temporal and neural expression profile suggests a general role in cell fate determination during nervous system differentiation.