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It is recognized that cell metabolism is tightly connected to other cellular processes such as regulation of gene expression. Metabolic pathways not only provide the precursor molecules necessary for gene expression, but they also provide ATP, the primary fuel driving gene expression. However, metabolic conditions are highly variable since nutrient uptake is not a uniform process. Thus, cells must continually calibrate gene expression to their changing metabolite and energy budgets. This review discusses recent advances in understanding the molecular and biophysical mechanisms that connect metabolism and gene regulation as cells navigate their growth, proliferation, and differentiation. Particular focus is given to these mechanisms in the context of organismal development. Gene expression may only account for 10% of a cell's energy budget, which undergoes complete ATP turnover every minute. Multilayered feedback repression enables gene expression to adapt to variable metabolic rates. Energy expenditure via nucleosome displacement and modification regulates transcription factor accessibility and ultrasensitivity. Aerobic glycolysis versus oxidative phosphorylation have distinct effects on epigenetic modifications. [ABSTRACT FROM AUTHOR]

Over the last decade, microRNAs have emerged as critical regulators in the expression and function of animal genomes. This review article discusses the relationship between microRNA-mediated regulation and the biology of the fruit fly Drosophila melanogaster . We focus on the roles that microRNAs play in tissue growth, germ cell development, hormone action, and the development and activity of the central nervous system. We also discuss the ways in which microRNAs affect robustness. Many gene regulatory networks are robust; they are relatively insensitive to the precise values of reaction constants and concentrations of molecules acting within the networks. MicroRNAs involved in robustness appear to be nonessential under uniform conditions used in conventional laboratory experiments. However, the robust functions of microRNAs can be revealed when environmental or genetic variation otherwise has an impact on developmental outcomes. [ABSTRACT FROM AUTHOR]

Yan is an ETS-domain transcription factor responsible for maintaining Drosophila eye cells in a multipotent state. Yan is at the core of a regulatory network that determines the time and place in which cells transit from multipotency to one of several differentiated lineages. Using a fluorescent reporter for Yan expression, we observed a biphasic distribution of Yan in multipotent cells, with a rapid inductive phase and slow decay phase. Transitions to various differentiated states occurred over the course of this dynamic process, suggesting that Yan expression level does not strongly determine cell potential. Consistent with this conclusion, perturbing Yan expression by varying gene dosage had no effect on cell fate transitions. However, we observed that as cells transited to differentiation, Yan expression became highly heterogeneous and this heterogeneity was transient. Signals received via the EGF Receptor were necessary for the transience in Yan noise since genetic loss caused sustained noise. Since these signals are essential for eye cells to differentiate, we suggest that dynamic heterogeneity of Yan is a necessary element of the transition process, and cell states are stabilized through noise reduction. [ABSTRACT FROM AUTHOR]

Robustness is a fundamental property of biological systems. The type of robustness that ensures uniform phenotypic outcomes in the face of variation during an organism's development is called canalization. Here, we discuss the roles that microRNAs play in providing canalization to animal development, citing recent theoretical and experimental advances. MicroRNAs repress protein expression, and they do this in ways that create thresholds in expression and provide adaptation to regulatory networks. Numerous examples have now been described where the developmental impact of environmental variation is suppressed by individual microRNAs. A recent paper has found that the impact of genomic variation between individuals is similarly suppressed by a microRNA operating in a developmental network. Thus, genetic variability is held in check, which is potentially important for both animal evolution and manifestation of disease. [ABSTRACT FROM AUTHOR]

Cells in the Drosophila retina have well-defined morphologies that are attained during tissue morphogenesis. We present a computer simulation of the epithelial tissue in which the global interfacial energy between cells is minimized. Experimental data for both normal cells and mutant cells either lacking or misexpressing the adhesion protein N-cadherin can be explained by a simple model incorporating salient features of morphogenesis that include the timing of N-cadherin expression in cells and its temporal relationship to the remodeling of cell-cell contacts. The simulations reproduce the geometries of wild-type and mutant cells, distinguish features of cadherin dynamics, and emphasize the importance of adhesion protein biogenesis and its timing with respect to cell remodeling. The simulations also indicate that N-cadherin protein is recycled from inactive interfaces to active interfaces, thereby modulating adhesion strengths between cells. [ABSTRACT FROM AUTHOR]

Background: Robustness to natural temperature fluctuations is critical to proper development in embryos and to cellular functions in adult organisms. However, mechanisms and pathways which govern temperature compensation remain largely unknown beyond circadian rhythms. Pathways which ensure robustness against temperature fluctuations may appear to be nonessential under favorable, uniform environmental conditions used in conventional laboratory experiments where there is little variation for which to compensate. The endo-siRNA pathway, which produces small double-stranded RNAs in Drosophila, appears to be nonessential for robust development of the embryo under ambient uniform temperature and to be necessary only for viral defense. Embryos lacking a functional endo-siRNA pathway develop into phenotypically normal adults. However, we hypothesized that small RNAs may regulate the embryo's response to temperature, as a ribonucleoprotein complex has been previously shown to mediate mammalian cell response to heat shock. Principal Findings: Here, we show that the genes DICER-2 and ARGONAUTE2, which code for integral protein components of the endo-siRNA pathway, are essential for robust development and temperature compensation in the Drosophila embryo when exposed to temperature perturbations. The regulatory functions of DICER-2 and ARGONAUTE2 were uncovered by using microfluidics to expose developing Drosophila embryos to a temperature step, in which each half of the embryo develops at a different temperature through developmental cycle 14. Under this temperature perturbation, dicer-2 or argonaute2 embryos displayed abnormal segmentation. The abnormalities in segmentation are presumably due to the inability of the embryo to compensate for temperature-induced differences in rate of development and to coordinate developmental timing in the anterior and posterior halves. A deregulation of the length of nuclear division cycles 10-14 is also observed in dicer-2 embryos at high temperatures. Conclusions: Results presented herein uncover a novel function of the endo-siRNA pathway in temperature compensation and cell cycle regulation, and we hypothesize that the endo-siRNA pathway may regulate the degradation of maternal cell cycle regulators. Endo-siRNAs may have a more general role buffering against environmental perturbations in other organisms. [ABSTRACT FROM AUTHOR]

Over the last decade, ∼20–30 nucleotide RNA molecules have emerged as critical regulators in the expression and function of eukaryotic genomes. Two primary categories of these small RNAs—short interfering RNAs (siRNAs) and microRNAs (miRNAs)—act in both somatic and germline lineages in a broad range of eukaryotic species to regulate endogenous genes and to defend the genome from invasive nucleic acids. Recent advances have revealed unexpected diversity in their biogenesis pathways and the regulatory mechanisms that they access. Our understanding of siRNA- and miRNA-based regulation has direct implications for fundamental biology as well as disease etiology and treatment. [Copyright &y& Elsevier]

Abstract: Lola is a transcription repressor that regulates axon guidance in the developing embryonic nervous system of Drosophila. Here, we show that Lola regulates two binary cell fate decisions guided by Notch inductive signaling in the developing eye: the R3–R4 and the R7–cone cell fate choices. Lola is required cell-autonomously in R3 for its specification, and Lola transforms R4 into R3 if overexpressed. Lola also promotes R7 fate at the expense of cone cell fate. Lola antagonizes Notch-dependent gene expression and Notch-dependent fate transformation. Expression analysis shows that Lola is constitutively present in all photoreceptors and cone cells. We propose that when a precursor cell receives a weak Notch inductive signal, it is not sufficiently strong to overcome the constitutive repression of target gene transcription by Lola. A precursor that receives a strong Notch inductive signal expresses target genes despite a constitutive repression by Lola. The predicted consequence of this mechanism is to sharpen a cell’s responsiveness to Notch signaling by creating a threshold. [Copyright &y& Elsevier]

The insect compound eye is one of the most precise and highly ordered patterns in the living world. It develops from an unpatterned simple epithelium by a series of cell fate decisions and complex morphogenetic movements. In the first days of metamorphosis, this interplay is particularly noticeable. Recent insights have revealed how interactions between neighboring cells drive the process. Interaction between Delta on cone cells and Notch proteins on the surface of their neighbors induces the first pigment cells to differentiate. The primary pigment cells then express a Nephrin protein, Hibris, that interacts with a different Nephrin, Roughest, on their neighbors. Heterophilic adhesion between Hibris and Roughest results in remodeling contacts between cells to favor their contact with the pigment cells. In conjunction, the primary pigment cells signal to their neighbors through the EGF receptor to survive, rather than undergo apoptosis. This sorting and culling process results in a sculpted pattern with a precise number and position of cells that is repeated hundreds of times in each compound eye. [Copyright &y& Elsevier]

Virus infection is generally disadvantageous to the host and strongly selects for host antiviral mechanisms. Therefore, viruses must develop counter-mechanisms to guarantee their survival. This arms race between pathogen and host leads to positive selection for both cellular antiviral mechanisms and viral inhibitors of such mechanisms. Here, we characterize this arms race in the context of the RNA interference (RNAi) pathway, which is used as an innate immune response against viral infection by animals. We review how RNAi is used as an antiviral strategy and the mechanisms that viruses have evolved to suppress the RNAi response. [Copyright &y& Elsevier]

The role of small RNAs as key regulators of mRNA turnover and translation has been well established. Recent advances indicate that the small RNAs termed microRNAs play important roles in animal development and physiology. Cellular activities such as proliferation, morphogenesis, apoptosis and differentiation are regulated by microRNAs. The expression of various genes are regulated by microRNAs, and several microRNAs act in reciprocal negative feedback loops with protein factors to control cell fate decisions that are triggered by signal transduction activity. These observations implicate small RNAs as important mediators of gene regulation in response to cell–cell signaling. The mechanism by which microRNAs silence gene expression is post-transcriptional, possibly influencing the stability, compartmentalization and translation of mRNAs. This mechanism is an efficient means to regulate production of a diverse range of proteins. [Copyright &y& Elsevier]

Summary: A critical question about signal transduction is how weak or transient activation of signaling pathways achieves a robust and long-term switch in gene expression. We report that a microRNA is part of a mechanism that makes cells sensitive to signals in the Drosophila eye. Expression of miR-7 is activated in cells as they begin differentiating into photoreceptors. This is dependent on EGF receptor (EGFR) signaling that triggers ERK-mediated degradation of the transcription factor Yan. In nonstimulated cells, Yan represses miR-7 transcription, whereas miR-7 RNA represses Yan protein expression in photoreceptors, by binding to sequences within its mRNA 3′UTR. We propose that reciprocal negative feedback between Yan and miR-7 ensures mutually exclusive expression, with Yan in progenitor cells and miR-7 in photoreceptor cells. Expression is switched when EGFR signaling transiently triggers Yan degradation. This two-tiered mechanism explains how signal transduction activity can robustly generate a stable change in gene-expression patterns. [Copyright &y& Elsevier]

The adherens junction functions to connect epithelial cells and maintain their polarized architecture. The geometry of the adherens junction, and consequently the shape of a cell, appears to reach an energetically favorable state. Cadherins within the adherens junction are necessary for cells to achieve this state. However, the view of an adherens junction as a static structure is at odds with the highly dynamic properties of epithelia during development. Interactions between the actin cytoskeleton and the adherens junction are required for certain cell shape changes. Recent insights into adherens junction remodeling have revealed the importance of polarized localization of myosin and Par3 at the adherens junction. [Copyright &y& Elsevier]

Although eukaryotic cells use RNA silencing to defend against transposons and pathogens, they also employ these pathways to regulate the expression of their own genes. Recent results have uncovered new examples of RNA silencing in controlling endogenous gene expression and have revealed novel mechanisms of downregulation. Bioinformatic analyses indicate that target genes in vertebrate species may number in the thousands. [Copyright &y& Elsevier]

Pattern formation of biological structures involves organizing different types of cells into a spatial configuration. In this study, we investigate the physical basis of biological patterning of the Drosophila retina in vivo. We demonstrate that E- and N-cadherins mediate apical adhesion between retina epithelial cells. Differential expression of N-cadherin within a sub-group of retinal cells (cone cells) causes them to form an overall shape that minimizes their surface contact with surrounding cells. The cells within this group, in both normal and experimentally manipulated conditions, pack together in the same way as soap bubbles do. The shaping of the cone cell group and packing of its components precisely imitate the physical tendency for surfaces to be minimized. Thus, simple patterned expression of N-cadherin results in a complex spatial pattern of cells owing to cellular surface mechanics. [ABSTRACT FROM AUTHOR]

The role of small RNAs as key regulators of mRNA turnover and translation has been well established. Recent advances indicate that the small RNAs termed microRNAs play important roles in cell proliferation, apoptosis and differentiation. Moreover, the microRNA mechanism is an efficient means to regulate production of a diverse range of proteins. As new microRNAs and their mRNA targets rapidly emerge, it is becoming apparent that RNA-based regulation of mRNAs may rival ubiquitination as a mechanism to control protein levels. [Copyright &y& Elsevier]

Double-stranded RNA induces sequence-specific inhibition of gene expression at a posttranscriptional level in eukaryotes (RNAi). This natural phenomenon has been developed into a tool for studying gene function in several model organisms, including Drosophila melanogaster. Transgenes bearing inverted repeats are able to exert an RNAi effect in Drosophila, but cloning difficulties and inconsistent silencing complicate the method. We have constructed a transgene containing inverted repeats separated by a functional intron such that mRNA produced by the transgene is predicted to form loopless hairpin RNA following splicing. A single copy of the transgene effectively and uniformly silences expression of a target gene (white) in transgenic flies. We have developed a vector that is designed to produce intron-spliced hairpin RNA corresponding to any Drosophila gene. The vector is under control of the upstream activating sequence (UAS) of the yeast transcriptional activator GAL4. The UAS/GAL4 system allows hairpin RNA to conditionally silence gene expression in Drosophila in a tissue-specific manner. Moreover, the presence of the intron spacer greatly enhances the stability of inverted-repeat sequences in bacteria, facilitating the cloning procedure. [Copyright &y& Elsevier]

The atypical cadherins Fat and Dachsous (Ds) signal through the Hippo pathway to regulate growth of numerous organs, including the Drosophila wing. Here, we find that Ds-Fat signaling tunes a unique feature of cell proliferation found to control the rate of wing growth during the third instar larval phase. The duration of the cell cycle increases in direct proportion to the size of the wing, leading to linear-like growth during the third instar. Ds-Fat signaling enhances the rate at which the cell cycle lengthens with wing size, thus diminishing the rate of wing growth. We show that this results in a complex but stereotyped relative scaling of wing growth with body growth in Drosophila. Finally, we examine the dynamics of Fat and Ds protein distribution in the wing, observing graded distributions that change during growth. However, the significance of these dynamics is unclear since perturbations in expression have negligible impact on wing growth. [ABSTRACT FROM AUTHOR]

Deals with a study that investigated the potential of double-stranded RNA to interfere with the function of genes in Drosophila. Background of the study; Experimental procedures; Results and discussion.

Studies the regulation of a system for transporting glucose in Stichococcus bacillaris. Transportation of glucose and related non-metabolized analogs into the cells of Stichococcus bacillaris; Stimulation of glucose transport capacity through incubation in medium of low osmotic medium; Implications on phycology.

Focuses on amino acids in the green alga Stichococcus bacillaris. Transport of glutamic and aspartic acids by carrier systems which obey saturation kinetics; Entry of the acidic amino acids by passive diffusion; Calculation of the Michaelis-Menten parameters.

Pattern formation of biological structures involves the arrangement of different types of cells in an ordered spatial configuration. In this study, we investigate the mechanism of patterning the Drosophila eye epithelium into a precise triangular grid of photoreceptor clusters called ommatidia. Previous studies had led to a long-standing biochemical model whereby a reaction-diffusion process is templated by recently formed ommatidia to propagate a molecular prepattern across the eye. Here, we find that the templating mechanism is instead, mechanochemical in origin; newly born columns of differentiating ommatidia serve as a template to spatially pattern flows that move epithelial cells into position to form each new column of ommatidia. Cell flow is generated by a source and sink, corresponding to narrow zones of cell dilation and contraction respectively, that straddle the growing wavefront of ommatidia. The newly formed lattice grid of ommatidia cells are immobile, deflecting, and focusing the flow of other cells. Thus, the self-organization of a regular pattern of cell fates in an epithelium is mechanically driven. [ABSTRACT FROM AUTHOR]

RNA-mediated interference (RNAi) is a recently discovered method to determine gene function in a number of organisms, including plants[SUP1], nematodes[SUP2], Drosophila[SUP3,4], zebrafish[SUP5], and mice[SUP6]. Injection of double-stranded RNA (dsRNA) corresponding to a single gene into organisms silences expression of the specific gene[SUP2-6]. Rapid degradation of mRNA in affected cells blocks gene expression[SUP2]. Despite the promise of RNAi as a tool for functional genomics, injection of dsRNA interferes with gene expression transiently and is not stably inherited[SUP2,3,5,6]. Consequently, use of RNAi to study gene function in the late stages of development has been limited. It is particularly problematic for development of disease models that reply on post-natal individuals. To circumvent this problem in Drosophila, we have developed a method to express dsRNA as an extended hairpin-loop RNA. This method has recently been successful in generating RNAi in the nematode Caenorhabditis elegans[SUP7]. The hairpin RNA is expressed from a transgene exhibiting dyad symmetry in a controlled temporal and spatial pattern. We report that the stably inherited transgene confers specific interference of gene expression in embryos, and tissues that give rise to adult structures such as the wings, legs, eyes, and brain. Thus, RNAi can be adapted to study late-acting gene function in Drosophila. The success of this approach in Drosophila and C. elegans suggests that a similar approach may prove useful to study gene function in higher organisms for which transgenic technology is available. [ABSTRACT FROM AUTHOR]

Invertebrate microRNAs (miRNAs) can suppress developmental variability that is caused by environmental and genetic variation. In this issue of Developmental Cell , Kasper et al. (2017) show that zebrafish miRNAs suppress variability in cardiovascular development during embryogenesis, providing insight into the conserved link between miRNAs and robustness. [ABSTRACT FROM AUTHOR]

Organismal development is a complex process, involving a vast number of molecular constituents interacting on multiple spatio-temporal scales in the formation of intricate body structures. Despite this complexity, development is remarkably reproducible and displays tolerance to both genetic and environmental perturbations. This robustness implies the existence of hidden simplicities in developmental programs. Here, using the Drosophila wing as a model system, we develop a new quantitative strategy that enables a robust description of biologically salient phenotypic variation. Analyzing natural phenotypic variation across a highly outbred population and variation generated by weak perturbations in genetic and environmental conditions, we observe a highly constrained set of wing phenotypes. Remarkably, the phenotypic variants can be described by a single integrated mode that corresponds to a non-intuitive combination of structural variations across the wing. This work demonstrates the presence of constraints that funnel environmental inputs and genetic variation into phenotypes stretched along a single axis in morphological space. Our results provide quantitative insights into the nature of robustness in complex forms while yet accommodating the potential for evolutionary variations. Methodologically, we introduce a general strategy for finding such invariances in other developmental contexts. [ABSTRACT FROM AUTHOR]

Gene expression is a regulated process fueled by ATP consumption. Therefore, regulation must be coupled to constraints imposed by the level of energy metabolism. Here, we explore this relationship both theoretically and experimentally. A stylized mathematical model predicts that activators of gene expression have variable impact depending on metabolic rate. Activators become less essential when metabolic rate is reduced and more essential when metabolic rate is enhanced. We find that, in the Drosophila eye, expression dynamics of the yan gene are less affected by loss of EGFR-mediated activation when metabolism is reduced, and the opposite effect is seen when metabolism is enhanced. The effects are also seen at the level of pattern regularity in the adult eye, where loss of EGFR-mediated activation is mitigated by lower metabolism. We propose that gene activation is tuned by energy metabolism to allow for faithful expression dynamics in the face of variable metabolic conditions. [ABSTRACT FROM AUTHOR]

Mosaic analysis provides a means to probe developmental processes in situ by generating loss-of-function mutants within otherwise wildtype tissues. Combining these techniques with quantitative microscopy enables researchers to rigorously compare RNA or protein expression across the resultant clones. However, visual inspection of mosaic tissues remains common in the literature because quantification demands considerable labor and computational expertise. Practitioners must segment cell membranes or cell nuclei from a tissue and annotate the clones before their data are suitable for analysis. Here, we introduce Fly-QMA, a computational framework that automates each of these tasks for confocal microscopy images of Drosophila imaginal discs. The framework includes an unsupervised annotation algorithm that incorporates spatial context to inform the genetic identity of each cell. We use a combination of real and synthetic validation data to survey the performance of the annotation algorithm across a broad range of conditions. By contributing our framework to the open-source software ecosystem, we aim to contribute to the current move toward automated quantitative analysis among developmental biologists. Author summary: Biologists use mosaic tissues to compare the behavior of genetically distinct cells within an otherwise equivalent context. The ensuing analysis is often limited to qualitative insight. However, it is becoming clear that quantitative models are needed to unravel the complexities of many biological systems. In this manuscript we introduce a computational framework that automates the quantification of mosaic analysis for Drosophila imaginal discs, a common setting for studies of developmental processes. The software extracts quantitative measurements from confocal images of mosaic tissues, rectifies any cross-talk between fluorescent reporters, and identifies clonally-related subpopulations of cells. Together, these functions allow users to rigorously ascribe changes in gene expression to the presence or absence of particular genes. We validate the performance of our framework using both real and synthetic data. We invite interested readers to apply these methods using our freely available software. [ABSTRACT FROM AUTHOR]

Aberrant microRNA (miRNA) expression correlates with human diseases such as cardiac disorders and cancer. Treatment of such disorders using miRNA-targeted therapeutics requires a thorough understanding of miRNA regulation in vivo. A recent paper in Nature by Davis et al. expands our understanding of miRNA biogenesis and maturation, elucidating a mechanism by which extracellular signaling directs cell differentiation via posttranscriptional regulation of miRNA expression. [Copyright &y& Elsevier]

The article reports on the discovery of the third class of small ribonucleic acid (RNA) species potential to genome control. The discovery of this type of small RNAs is linked to the association of certain Argonaute proteins with messenger RNAs and small interfering RNAs to form ribonucleoprotein complexes that repress the expression of target messenger RNAs. Also, the Piwi protein of Drosophila melanogaster plays an important role in germline development, and that it is essential for spermatogenesis.

This article focuses on diseases in historical periods. In Sophocles' Oedipus the King, the citizens of Thebes are cursed by a plague that will end only when the murderer of Laius, their former ruler, is identified and banished. Although there are many things to be learned from this drama, one conclusion relates directly to the process of discovery: The object of an intensive search sometimes turns out to be hiding in plain view. The Hannon and Joshua-Tor laboratories have now used a battery of biophysical, biochemical, and genetic approaches to examine Argonaute structure and function 1, 2.

Provides information on the roles that nucleases play in both making and destroying RNA associated with small RNA. Overview of targeting messenger RNA transcripts with complementary sequence for rapid destruction; Ways that small RNA silence gene expression; Depletion of protein levels in Caenorhabditis elegans.

Cell state transitions are often triggered by large changes in the concentrations of transcription factors and therefore large differences in their stoichiometric ratios. Whether cells can elicit transitions using modest changes in the ratios of co-expressed factors is unclear. Here, we investigate how cells in the Drosophila eye resolve state transitions by quantifying the expression dynamics of the ETS transcription factors Pnt and Yan. Eye progenitor cells maintain a relatively constant ratio of Pnt/Yan protein, despite expressing both proteins with pulsatile dynamics. A rapid and sustained twofold increase in the Pnt/Yan ratio accompanies transitions to photoreceptor fates. Genetic perturbations that modestly disrupt the Pnt/Yan ratio produce fate transition defects consistent with the hypothesis that transitions are normally driven by a twofold shift in the ratio.A biophysicalmodel based on cooperative Yan-DNA binding coupled with non-cooperative Pnt-DNA binding illustrates how twofold ratio changes could generate ultrasensitive changes in target gene transcription to drive fate transitions. Thus, coupling cell state transitions to the Pnt/Yan ratio sensitizes the system to modest fold-changes, conferring robustness and ultrasensitivity to the developmental program. [ABSTRACT FROM AUTHOR]

Fragile X syndrome is caused by loss of expression of FMRP, a protein proposed to act as a regulator of mRNA translation which promotes synaptic maturation and function. Now FMRP has been found to associate with the RNP complex that mediates post-transcriptional silencing by RNAi. [Copyright &y& Elsevier]

Genetic variation is prevalent among individuals of the same species and yet the potential effects of genetic variation on developmental outcomes are frequently suppressed. Understanding the mechanisms that are responsible for this suppression is an important goal. Previously, we found that the microRNA miR-9a mitigates the impact of natural genetic variants that promote the development of scutellar bristles in adult Drosophila. Here we find that miR-9a does not affect the impact of genetic variants that inhibit the development of scutellar bristles. We show this using both directional and stabilizing selection in the laboratory. This specificity of action suggests that miR-9a does not interact with all functional classes of developmental genetic variants affecting sensory organ development. We also investigate the impact of miR-9a on a fitness trait, which is adult viability. At elevated physiological temperatures, miR-9a contributes to viability through masking genetic variants that hinder adult viability. We conclude that miR-9a activity in different developmental networks contributes to suppression of natural variants from perturbing development. [ABSTRACT FROM AUTHOR]

Summary: Because microRNAs (miRNAs) influence the expression of many genes in cells, discovering how the miRNA pathway is regulated is an important area of investigation. We found that the Drosophila miRNA-induced silencing complex (miRISC) exists in multiple forms. A constitutive form, called G-miRISC, is comprised of Ago1, miRNA, and GW182. Two distinct miRISC complexes that lack GW182 are regulated by mitogenic signaling. Exposure of cells to serum, lipids, or the tumor promoter PMA suppressed formation of these complexes. P-miRISC is comprised of Ago1, miRNA, and Loqs-PB, and it associates with mRNAs assembled into polysomes. The other regulated Ago1 complex associates with membranous organelles and is likely an intermediate in miRISC recycling. The formation of these complexes is correlated with a 5- to 10-fold stronger repression of target gene expression inside cells. Taken together, these results indicate that mitogenic signaling regulates the miRNA effector machinery to attenuate its repressive activities. [Copyright &y& Elsevier]

In multicellular organisms, cells pack together to form tissues of intricate and welt defined morphology. How such cell-packing geometries arise is an important open question in biology, because the functionality of many differentiated tissues depends on their reliable formation. We show that combining adhesive forces due to E- and N-cadherin with a quantitative description of cell membrane elasticity in an interfacial energy model explains not only the qualitative neighbor relations, but also the detailed geometry of a tissue. The characteristic cellular geometries in the eyes of both wild-type Drosophila and genetic mutants are accurately reproduced by using a fixed set of few, physically motivated parameters. The model predicts adhesion strengths in the eye epithelium, quantifies their role relative to membrane elasticity, and reveals how simple minimization of interfacial energy can give rise to complex geometric patterns of important biological functionality. [ABSTRACT FROM AUTHOR]

Spherical nucleic acids (SNAs) are nanostructures formed by chemically conjugating short linear strands of oligonucleotides to a nanoparticle template. When made with modified small interfering RNA (siRNA) duplexes, SNAs act as single-entity transfection and gene silencing agents and have been used as lead therapeutic constructs in several disease models. However, the manner in which modified siRNA duplex strands that comprise the SNA lead to gene silencing is not understood. Herein, a systematic analysis of siRNA biochemistry involving SNAs shows that Dicer cleaves the modified siRNA duplex from the surface of the nanoparticle, and the liberated siRNA subsequently functions in a way that is dependent on the canonical RNA interference mechanism. By leveraging this understanding, a class of SNAs was chemically designed which increases the siRNA content by an order of magnitude through covalent attachment of each strand of the duplex. As a consequence of increased nucleic acid content, this nanostructure architecture exhibits less cell cytotoxicity than conventional SNAs without a decrease in siRNA activity. [ABSTRACT FROM AUTHOR]

A response by Jian Lu, Yang Shen, Richard W. Carthew, San Ming Wang and Chung-I Wu to a letter to the editor about their article "Evolutionary flux of canonical microRNAs and mirtrons in Drosophila," in the January 2010 issue is presented.

Transposable elements (TEs) are silenced in germ cells by a mechanism in which PIWI proteins generate and use PIWIinteracting ribonucleic acid (piRNA) to repress expression of TE genes. piRNA biogenesis occurs by an amplification cycle in microscopic organelles called nuage granules, which are localized to the outer face of the nuclear envelope. One cofactor required for amplification is the helicase Spindle-E (Spn-E). We found that the Spn-E protein physically associates with the Tudor domain protein Qin and the PIWI proteins Aubergine (Aub) and Argonaute3 (Ago3). Spn-E and Qin proteins are mutually dependent for their exit from nuage granules, whereas Spn-E and both Aub and Ago3 are mutually dependent for their entry or retention in nuage. The result is a dynamic cycling of Spn-E and its associated factors in and out of nuage granules. This implies that nuage granules can be considered to be hubs for active, mobile, and transient complexes. We suggest that this is in some way coupled with the execution of the piRNA amplification cycle. [ABSTRACT FROM AUTHOR]

Abstract: The biochemical regulatory network downstream of receptor tyrosine kinase (RTK) signaling is controlled by two opposing ETS family members: the transcriptional activator Pointed (Pnt) and the transcriptional repressor Yan. A bistable switch model has been invoked to explain how pathway activation can drive differentiation by shifting the system from a high-Yan/low-Pnt activity state to a low-Yan/high-Pnt activity state. Although the model explains yan and pnt loss-of-function phenotypes in several different cell types, how Yan and Pointed protein expression dynamics contribute to these and other developmental transitions remains poorly understood. Toward this goal we have used a functional GFP-tagged Pnt transgene (Pnt-GFP) to perform a comparative study of Yan and Pnt protein expression throughout Drosophila development. Consistent with the prevailing model of the Pnt–Yan network, we found numerous instances where Pnt–GFP and Yan adopt a mutually exclusive pattern of expression. However we also observed many examples of co-expression. While some co-expression occurred in cells where RTK signaling is presumed low, other co-expression occurred in cells with high RTK signaling. The instances of co-expressed Yan and Pnt-GFP in tissues with high RTK signaling cannot be explained by the current model, and thus they provide important contexts for future investigation of how context-specific differences in RTK signaling, network topology, or responsiveness to other signaling inputs, affect the transcriptional response. [Copyright &y& Elsevier]

Summary: Gene expression has to withstand stochastic, environmental, and genomic perturbations. For example, in the latter case, 0.5%–1% of the human genome is typically variable between any two unrelated individuals. Such diversity might create problematic variability in the activity of gene regulatory networks and, ultimately, in cell behaviors. Using multigenerational selection experiments, we find that for the Drosophila proneural network, the effect of genomic diversity is dampened by miR-9a-mediated regulation of senseless expression. Reducing miR-9a regulation of the Senseless transcription factor frees the genomic landscape to exert greater phenotypic influence. Whole-genome sequencing identified genomic loci that potentially exert such effects. A larger set of sequence variants, including variants within proneural network genes, exhibits these characteristics when miR-9a concentration is reduced. These findings reveal that microRNA-target interactions may be a key mechanism by which the impact of genomic diversity on cell behavior is dampened. [ABSTRACT FROM AUTHOR]

In Drosophila, post-transcriptional gene silencing occurs when exogenous or endogenous double stranded RNA (dsRNA) is processed into small interfering RNAs (siRNAs) by Dicer-2 (Dcr-2) in association with a dsRNA-binding protein (dsRBP) cofactor called Loquacious (Loqs-PD). siRNAs are then loaded onto Argonaute-2 (Ago2) by the action of Dcr-2 with another dsRBP cofactor called R2D2. Loaded Ago2 executes the destruction of target RNAs that have sequence complementarity to siRNAs. Although Dcr-2, R2D2, and Ago2 are essential for innate antiviral defense, the mechanism of virus-derived siRNA (vsiRNA) biogenesis and viral target inhibition remains unclear. Here, we characterize the response mechanism mediated by siRNAs against two different RNA viruses that infect Drosophila. In both cases, we show that vsiRNAs are generated by Dcr-2 processing of dsRNA formed during viral genome replication and, to a lesser extent, viral transcription. These vsiRNAs seem to preferentially target viral polyadenylated RNA to inhibit viral replication. Loqs-PD is completely dispensable for silencing of the viruses, in contrast to its role in silencing endogenous targets. Biogenesis of vsiRNAs is independent of both Loqs-PD and R2D2. R2D2, however, is required for sorting and loading of vsiRNAs onto Ago2 and inhibition of viral RNA expression. Direct injection of viral RNA into Drosophila results in replication that is also independent of Loqs-PD. This suggests that triggering of the antiviral pathway is not related to viral mode of entry but recognition of intrinsic features of virus RNA. Our results indicate the existence of a vsiRNA pathway that is separate from the endogenous siRNA pathway and is specifically triggered by virus RNA. We speculate that this unique framework might be necessary for a prompt and efficient antiviral response. [ABSTRACT FROM AUTHOR]

ETS family transcription factors are evolutionarily conserved downstream effectors of Ras/MAPK signaling with critical roles in development and cancer. In Drosophila, the ETS repressor Yan regulates cell proliferation and differentiation in a variety of tissues; however, the mechanisms of Yan-mediated repression are not well understood and only a few direct target genes have been identified. Yan, like its human ortholog TEL1, self-associates through an N-terminal sterile α-motif (SAM), leading to speculation that Yan/TEL1 polymers may spread along chromatin to form large repressive domains. To test this hypothesis, we created a monomeric form of Yan by recombineering a point mutation that blocks SAM-mediated self-association into the yan genomic locus and compared its genome-wide chromatin occupancy profile to that of endogenous wild-type Yan. Consistent with the spreading model predictions, wild-type Yan-bound regions span multiple kilobases. Extended occupancy patterns appear most prominent at genes encoding crucial developmental regulators and signaling molecules and are highly conserved between Drosophila melanogaster and D. virilis, suggesting functional relevance. Surprisingly, although occupancy is reduced, the Yan monomer still makes extensive multikilobase contacts with chromatin, with an overall pattern similar to that of wild-type Yan. Despite its near-normal chromatin recruitment, the repressive function of the Yan monomer is significantly impaired, as evidenced by elevated target gene expression and failure to rescue a yan null mutation. Together our data argue that SAM-mediated polymerization contributes to the functional output of the active Yan repressive complexes that assemble across extended stretches of chromatin, but does not directly mediate recruitment to DNA or chromatin spreading. [ABSTRACT FROM AUTHOR]

In Drosophila, Notch and Egfr signaling regulate the determination of many cell types, and yet how these common signals generate cell-specific transcription is not well understood. In the compound eye, prospero (pros) is transcribed specifically in R7 photoreceptors and cone cells. We show that the transcription of pros is activated by two visual-specific transcription selectors, Glass and Sine Oculis, that bind to an enhancer and promote its activation. Together with the pre-patterning transcription factor Lozenge, these factors work in a highly combinatorial manner, such that cells missing any one factor transcribe pros only weakly, if at all. However, the factors are not sufficient to activate the enhancer because of an additional requirement for both Notch and Egfr signals. The loss of Notch signaling produces a `salt and pepper' effect, with some cells expressing near-normal levels and others expressing no detectable pros at all; thus, the signaling loss does not produce a uniformly reduced level of transcription activity in cells. This suggests a probabilistic mechanism, in which Notch signals influence the likelihood that the enhancer is inactive or fully active in any given cell. The activity level, therefore, is dictated by the proper combination of highly cooperative selector and pre-pattern factors present in the cell. [ABSTRACT FROM AUTHOR]

RNA silencing (RNAI) induced by virus-derived double-stranded RNA (dsRNA), which is in a sense regarded as a pathogen-associated molecular pattern (PAMP) of viruses, is a general plant defense mechanism. To counteract this defense, plant viruses express RNA silencing suppressors (RSSs), many of which bind to dsRNA and attenuate RNAi. We showed that the tobacco calmodulin-like protein, rgs-CaM, counterattacked viral RSSs by binding to their dsRNA-binding domains and sequestering them from inhibiting RNAi. Autophagy-like protein degradation seemed to operate to degrade RSSs with the sacrifice of rgs-CaM. These RSSs could thus be regarded as secondary viral PAMPs. This study uncovered a unique defense system in which an rgs-CaM-mediated countermeasure against viral RSSs enhanced host antiviral RNAi in tobacco. [ABSTRACT FROM AUTHOR]

Mammals lacking BLOC-3 have impaired formation of melanosomes, a type of lysosome-related organelle (LRO), and, in earlier work, we found that a subunit of the BLOC-3 complex inhibits loading of Argonaute (Ago) proteins with small ribonucleic acids (RNAs) in Drosophila melanogaster cells. Small RNAs such as small interfering RNAs (siRNAs) direct Ago proteins to repress the stability of messenger RNA transcripts. In this paper, we show that BLOC-3 is required for biogenesis of Drosophila LROs called pigment granules. Other complexes that sort cargo to pigment LROs also negatively regulate siRNA activity. However, regulation is not obligately linked to biogenesis of LROs but instead to specific cargo-sorting processes. Negative regulation is also not linked to sorting into all LROs but only a specific class of pigment LRO. Thus, regulation of siRNA activity is tied to sorting of specific types of cargo to particular LROs. [ABSTRACT FROM AUTHOR]

Mammals lacking BLOC-3 have impaired formation of melanosomes, a type of lysosome-related Morganelle (LRO), and, in earlier work, we found that a subunit of the BLOC-3 complex inhibits loading of Argonaute (Ago) proteins with small ribonucleic acids (RNAs) in Drosophila melanogaster cells. Small RNAs such as small interfering RNAs (siRNAs) direct Ago proteins to repress the stability of messenger RNA transcripts. In this paper , we show that BLOC-3 is required for biogenesis of Drosophila LROs called pigment granules. Other complexes that sort cargo to pigment LROs also negatively regulate siRNA activity. However , regulation is not obligately linked to biogenesis of LROs but instead to specific cargo-sorting processes. Negative regulation is also not linked to sorting into all LROs but only a specific class of pigment LRO. Thus, regulation of siRNA activity is tied to sorting of specific types of cargo to particular LROs. [ABSTRACT FROM AUTHOR]

The Drosophila protein kinase Par-1 is expressed throughout Drosophila development, but its function has not been extensively characterized because of oocyte lethality of null mutants. In this report, we have characterized the function of Par-1 in embryonic and post-embryonic epithelia. Par-1 protein is dynamically localized during embryonic cell polarization, transiently restricted to the lateral membrane domain, followed by apicolateral localization. We depleted maternal and zygotic par-1 by RNAi and revealed a requirement for Par-1 in establishing cell polarity. Par-1 restricts the coalescing adherens junction to an apicolateral position and prevents its widespread formation along the lateral domain. Par-1 also promotes the localization of lateral membrane proteins such as Delta. These activities are important for the further development of cell polarity during gastrulation. By contrast, Par-1 is not essential to maintain epithelial polarity once it has been established. However, it still has a maintenance role since overexpression causes severe polarity disruption. Additionally, we find a novel role for Par-1 in Notch signal transduction during embryonic neurogenesis and retina determination. Epistasis analysis indicates that Par-1 functions upstream of Notch and is critical for proper localization of the Notch ligand Delta. [ABSTRACT FROM AUTHOR]