Hedgehog Signaling Strength Is Orchestrated by the mir-310 Cluster of MicroRNAs in Response to Diet

Since the discovery of microRNAs (miRNAs) only two decades ago, they have emerged as an essential component of the gene regulatory machinery. miRNAs have seemingly paradoxical features: a single miRNA is able to simultaneously target hundreds of genes, while its presence is mostly dispensable for animal viability under normal conditions. It is known that miRNAs act as stress response factors; however, it remains challenging to determine their relevant targets and the conditions under which they function. To address this challenge, we propose a new workflow for miRNA function analysis, by which we found that the evolutionarily young miRNA family, the mir-310s (mir-310/mir-311/mir-312/mir-313), are important regulators of Drosophila metabolic status. mir-310s-deficient animals have an abnormal diet-dependent expression profile for numerous diet-sensitive components, accumulate fats, and show various physiological defects. We found that the mir-310s simultaneously repress the production of several regulatory factors (Rab23, DHR96, and Ttk) of the evolutionarily conserved Hedgehog (Hh) pathway to sharpen dietary response. As the mir-310s expression is highly dynamic and nutrition sensitive, this signal relay model helps to explain the molecular mechanism governing quick and robust Hh signaling responses to nutritional changes. Additionally, we discovered a new component of the Hh signaling pathway in Drosophila, Rab23, which cell autonomously regulates Hh ligand trafficking in the germline stem cell niche. How organisms adjust to dietary fluctuations to sustain healthy homeostasis is an intriguing research topic. These data are the first to report that miRNAs can act as executives that transduce nutritional signals to an essential signaling pathway. This suggests miRNAs as plausible therapeutic agents that can be used in combination with low calorie and cholesterol diets to manage quick and precise tissue-specific responses to nutritional changes.     

Neprilysins: An Evolutionarily Conserved Family of Metalloproteases That Play Important Roles in Reproduction in Drosophila

Members of the M13 class of metalloproteases have been implicated in diseases and in reproductive fitness. Nevertheless, their physiological role remains poorly understood. To obtain a tractable model with which to analyze this protein family’s function, we characterized the gene family in Drosophila melanogaster and focused on reproductive phenotypes. The D. melanogaster genome contains 24 M13 class protease homologs, some of which are orthologs of human proteases, including neprilysin. Many are expressed in the reproductive tracts of either sex. Using RNAi we individually targeted the five Nep genes most closely related to vertebrate neprilysin, Nep1-5, to investigate their roles in reproduction. A reduction in Nep1, Nep2, or Nep4 expression in females reduced egg laying. Nep1 and Nep2 are required in the CNS and the spermathecae for wild-type fecundity. Females that are null for Nep2 also show defects as hosts of sperm competition as well as an increased rate of depletion for stored sperm. Furthermore, eggs laid by Nep2 mutant females are fertilized normally, but arrest early in embryonic development. In the male, only Nep1 was required to induce normal patterns of female egg laying. Reduction in the expression of Nep2-5 in the male did not cause any dramatic effects on reproductive fitness, which suggests that these genes are either nonessential for male fertility or perform redundant functions. Our results suggest that, consistent with the functions of neprilysins in mammals, these proteins are also required for reproduction in Drosophila, opening up this model system for further functional analysis of this protein class and their substrates.     

The Drosophila wings apart Gene Anchors a Novel,Evolutionarily Conserved Pathway of Neuromuscular Development

wings apart (wap) is a recessive, semilethal gene located on the X chromosome in Drosophila melanogaster, which is required for normal wing-vein patterning. We show that the wap mutation also results in loss of the adult jump muscle. We use complementation mapping and gene-specific RNA interference to localize the wap locus to the proximal X chromosome. We identify the annotated gene CG14614 as the gene affected by the wap mutation, since one wap allele contains a non-sense mutation in CG14614, and a genomic fragment containing only CG14614 rescues the jump-muscle phenotypes of two wap mutant alleles. The wap gene lies centromere-proximal to touch-insensitive larva B and centromere-distal to CG14619, which is tentatively assigned as the gene affected in introverted mutants. In mutant wap animals, founder cell precursors for the jump muscle are specified early in development, but are later lost. Through tissue-specific knockdowns, we demonstrate that wap function is required in both the musculature and the nervous system for normal jump-muscle formation. wap/CG14614 is homologous to vertebrate wdr68, DDB1 and CUL4 associated factor 7, which also are expressed in neuromuscular tissues. Thus, our findings provide insight into mechanisms of neuromuscular development in higher animals and facilitate the understanding of neuromuscular diseases that may result from mis-expression of muscle-specific or neuron-specific genes.     

A Novel Gene Controlling the Timing of Courtship Initiation in Drosophila melanogaster

Over the past 35 years, developmental geneticists have made impressive progress toward an understanding of how genes specify morphology and function, particularly as they relate to the specification of each physical component of an organism. In the last 20 years, male courtship behavior in Drosophila melanogaster has emerged as a robust model system for the study of genetic specification of behavior. Courtship behavior is both complex and innate, and a single gene, fruitless (fru), is both necessary and sufficient for all aspects of the courtship ritual. Typically, loss of male-specific Fruitless protein function results in male flies that perform the courtship ritual incorrectly, slowly, or not at all. Here we describe a novel requirement for fru: we have identified a group of cells in which male Fru proteins are required to reduce the speed of courtship initiation. In addition, we have identified a gene, Trapped in endoderm 1 (Tre1), which is required in these cells for normal courtship and mating behavior. Tre1 encodes a G-protein-coupled receptor required for establishment of cell polarity and cell migration and has previously not been shown to be involved in courtship behavior. We describe the results of feminization of the Tre1-expressing neurons, as well as the effects on courtship behavior of mutation of Tre1. In addition, we show that Tre1 is expressed in a sexually dimorphic pattern in the central and peripheral nervous systems and investigate the role of the Tre1 cells in mate identification.     

Class I Myosins Have Overlapping and Specialized Functions in Left-Right Asymmetric Development in Drosophila

The class I myosin genes are conserved in diverse organisms, and their gene products are involved in actin dynamics, endocytosis, and signal transduction. Drosophila melanogaster has three class I myosin genes, Myosin 31DF (Myo31DF), Myosin 61F (Myo61F), and Myosin 95E (Myo95E). Myo31DF, Myo61F, and Myo95E belong to the Myosin ID, Myosin IC, and Myosin IB families, respectively. Previous loss-of-function analyses of Myo31DF and Myo61F revealed important roles in left–right (LR) asymmetric development and enterocyte maintenance, respectively. However, it was difficult to elucidate their roles in vivo, because of potential redundant activities. Here we generated class I myosin double and triple mutants to address this issue. We found that the triple mutant was viable and fertile, indicating that all three class I myosins were dispensable for survival. A loss-of-function analysis revealed further that Myo31DF and Myo61F, but not Myo95E, had redundant functions in promoting the dextral LR asymmetric development of the male genitalia. Myo61F overexpression is known to antagonize the dextral activity of Myo31DF in various Drosophila organs. Thus, the LR-reversing activity of overexpressed Myo61F may not reflect its physiological function. The endogenous activity of Myo61F in promoting dextral LR asymmetric development was observed in the male genitalia, but not the embryonic gut, another LR asymmetric organ. Thus, Myo61F and Myo31DF, but not Myo95E, play tissue-specific, redundant roles in LR asymmetric development. Our studies also revealed differential colocalization of the class I myosins with filamentous (F)-actin in the brush border of intestinal enterocytes.     

Mutations to the piRNA Pathway Component Aubergine Enhance Meiotic Drive of Segregation Distorter in Drosophila melanogaster

Diploid sexual reproduction involves segregation of allelic pairs, ensuring equal representation of genotypes in the gamete pool. Some genes, however, are able to “cheat” the system by promoting their own transmission. The Segregation distorter (Sd) locus in Drosophila melanogaster males is one of the best-studied examples of this type of phenomenon. In this system the presence of Sd on one copy of chromosome 2 results in dysfunction of the non–Sd-bearing (Sd⁺) sperm and almost exclusive transmission of Sd to the next generation. The mechanism by which Sd wreaks such selective havoc has remained elusive. However, its effect requires a target locus on chromosome 2 known as Responder (Rsp). The Rsp locus comprises repeated copies of a satellite DNA sequence and Rsp copy number correlates with sensitivity to Sd. Under distorting conditions during spermatogenesis, nuclei with chromosomes containing greater than several hundred Rsp repeats fail to condense chromatin and are eliminated. Recently, Rsp sequences were found as small RNAs in association with Argonaute family proteins Aubergine (Aub) and Argonaute3 (AGO3). These proteins are involved in a germline-specific RNAi mechanism known as the Piwi-interacting RNA (piRNA) pathway, which specifically suppresses transposon activation in the germline. Here, we evaluate the role of piRNAs in segregation distortion by testing the effects of mutations to piRNA pathway components on distortion. Further, we specifically targeted mutations to the aub locus of a Segregation Distorter (SD) chromosome, using ends-out homologous recombination. The data herein demonstrate that mutations to piRNA pathway components act as enhancers of SD.     

Chromatin-Associated Proteins HP1 and Mod(mdg4) Modify Y-Linked Regulatory Variation in the Drosophila Testis

Chromatin remodeling is crucial for gene regulation. Remodeling is often mediated through chemical modifications of the DNA template, DNA-associated proteins, and RNA-mediated processes. Y-linked regulatory variation (YRV) refers to the quantitative effects that polymorphic tracts of Y-linked chromatin exert on gene expression of X-linked and autosomal genes. Here we show that naturally occurring polymorphisms in the Drosophila melanogaster Y chromosome contribute disproportionally to gene expression variation in the testis. The variation is dependent on wild-type expression levels of mod(mdg4) as well as Su(var)205; the latter gene codes for heterochromatin protein 1 (HP1) in Drosophila. Testis-specific YRV is abolished in genotypes with heterozygous loss-of-function mutations for mod(mdg4) and Su(var)205 but not in similar experiments with JIL-1. Furthermore, the Y chromosome differentially regulates several ubiquitously expressed genes. The results highlight the requirement for wild-type dosage of Su(var)205 and mod(mdg4) in enabling naturally occurring Y-linked regulatory variation in the testis. The phenotypes that emerge in the context of wild-type levels of the HP1 and Mod(mdg4) proteins might be part of an adaptive response to the environment.     

Identification of Mob2, a Novel Regulator of Larval Neuromuscular Junction Morphology,in Natural Populations of Drosophila melanogaster

Although evolutionary changes must take place in neural connectivity and synaptic architecture as nervous systems become more complex, we lack understanding of the general principles and specific mechanisms by which these changes occur. Previously, we found that morphology of the larval neuromuscular junction (NMJ) varies extensively among different species of Drosophila but is relatively conserved within a species. To identify specific genes as candidates that might underlie phenotypic differences in NMJ morphology among Drosophila species, we performed a genetic analysis on one of two phenotypic variants we found among 20 natural isolates of Drosophila melanogaster. We discovered genetic polymorphisms for both positive and negative regulators of NMJ growth segregating within the variant line. Focusing on one subline, that displayed NMJ overgrowth, we mapped the phenotype to Mob2 [Monopolar spindle (Mps) one binding protein 2)], a gene encoding a Nuclear Dbf2 (Dumbbell formation 2)-Related (NDR) kinase activator. We confirmed this identification by transformation rescue experiments and showed that presynaptic expression of Mob2 is necessary and sufficient to regulate NMJ growth. Mob2 interacts in a dominant, dose-dependent manner with tricornered but not with warts, to cause NMJ overgrowth, suggesting that Mob2 specifically functions in combination with the former NDR kinase to regulate NMJ development. These results demonstrate the feasibility and utility of identifying genetic variants affecting NMJ morphology in natural populations of Drosophila. These variants can lead to discovery of new genes and molecular mechanisms that regulate NMJ development while also providing new information that can advance our understanding of mechanisms that underlie nervous system evolution.     

The Mps1 Kinase Modulates the Recruitment and Activity of Cnn1CENP-T at Saccharomyces cerevisiae Kinetochores

Kinetochores are conserved protein complexes that bind the replicated chromosomes to the mitotic spindle and then direct their segregation. To better comprehend Saccharomyces cerevisiae kinetochore function, we dissected the phospho-regulated dynamic interaction between conserved kinetochore protein Cnn1^CENP-T, the centromere region, and the Ndc80 complex through the cell cycle. Cnn1 localizes to kinetochores at basal levels from G1 through metaphase but accumulates abruptly at anaphase onset. How Cnn1 is recruited and which activities regulate its dynamic localization are unclear. We show that Cnn1 harbors two kinetochore-localization activities: a C-terminal histone-fold domain (HFD) that associates with the centromere region and a N-terminal Spc24/Spc25 interaction sequence that mediates linkage to the microtubule-binding Ndc80 complex. We demonstrate that the established Ndc80 binding site in the N terminus of Cnn1, Cnn1^60–84, should be extended with flanking residues, Cnn1^25–91, to allow near maximal binding affinity to Ndc80. Cnn1 localization was proposed to depend on Mps1 kinase activity at Cnn1–S74, based on in vitro experiments demonstrating the Cnn1–Ndc80 complex interaction. We demonstrate that from G1 through metaphase, Cnn1 localizes via both its HFD and N-terminal Spc24/Spc25 interaction sequence, and deletion or mutation of either region results in anomalous Cnn1 kinetochore levels. At anaphase onset (when Mps1 activity decreases) Cnn1 becomes enriched mainly via the N-terminal Spc24/Spc25 interaction sequence. In sum, we provide the first in vivo evidence of Cnn1 preanaphase linkages with the kinetochore and enrichment of the linkages during anaphase.     

New Regulators of Clathrin-Mediated Endocytosis Identified in Saccharomyces cerevisiae by Systematic Quantitative Fluorescence Microscopy

Despite the importance of clathrin-mediated endocytosis (CME) for cell biology, it is unclear if all components of the machinery have been discovered and many regulatory aspects remain poorly understood. Here, using Saccharomyces cerevisiae and a fluorescence microscopy screening approach we identify previously unknown regulatory factors of the endocytic machinery. We further studied the top scoring protein identified in the screen, Ubx3, a member of the conserved ubiquitin regulatory X (UBX) protein family. In vivo and in vitro approaches demonstrate that Ubx3 is a new coat component. Ubx3-GFP has typical endocytic coat protein dynamics with a patch lifetime of 45 ± 3 sec. Ubx3 contains a W-box that mediates physical interaction with clathrin and Ubx3-GFP patch lifetime depends on clathrin. Deletion of the UBX3 gene caused defects in the uptake of Lucifer Yellow and the methionine transporter Mup1 demonstrating that Ubx3 is needed for efficient endocytosis. Further, the UBX domain is required both for localization and function of Ubx3 at endocytic sites. Mechanistically, Ubx3 regulates dynamics and patch lifetime of the early arriving protein Ede1 but not later arriving coat proteins or actin assembly. Conversely, Ede1 regulates the patch lifetime of Ubx3. Ubx3 likely regulates CME via the AAA-ATPase Cdc48, a ubiquitin-editing complex. Our results uncovered new components of the CME machinery that regulate this fundamental process.     

Identification of Genes That Promote or Inhibit Olfactory Memory Formation in Drosophila

Genetic screens in Drosophila melanogaster and other organisms have been pursued to filter the genome for genetic functions important for memory formation. Such screens have employed primarily chemical or transposon-mediated mutagenesis and have identified numerous mutants including classical memory mutants, dunce and rutabaga. Here, we report the results of a large screen using panneuronal RNAi expression to identify additional genes critical for memory formation. We identified >500 genes that compromise memory when inhibited (low hits), either by disrupting the development and normal function of the adult animal or by participating in the neurophysiological mechanisms underlying memory formation. We also identified >40 genes that enhance memory when inhibited (high hits). The dunce gene was identified as one of the low hits and further experiments were performed to map the effects of the dunce RNAi to the α/β and γ mushroom body neurons. Additional behavioral experiments suggest that dunce knockdown in the mushroom body neurons impairs memory without significantly affecting acquisition. We also characterized one high hit, sickie, to show that RNAi knockdown of this gene enhances memory through effects in dopaminergic neurons without apparent effects on acquisition. These studies further our understanding of two genes involved in memory formation, provide a valuable list of genes that impair memory that may be important for understanding the neurophysiology of memory or neurodevelopmental disorders, and offer a new resource of memory suppressor genes that will aid in understanding restraint mechanisms employed by the brain to optimize resources.     

Redundant Regulation of Cdk1 Tyrosine Dephosphorylation in Saccharomyces cerevisiae

Cdk1 activity drives both mitotic entry and the metaphase-to-anaphase transition in all eukaryotes. The kinase Wee1 and the phosphatase Cdc25 regulate the mitotic activity of Cdk1 by the reversible phosphorylation of a conserved tyrosine residue. Mutation of cdc25 in Schizosaccharomyces pombe blocks Cdk1 dephosphorylation and causes cell cycle arrest. In contrast, deletion of MIH1, the cdc25 homolog in Saccharomyces cerevisiae, is viable. Although Cdk1-Y19 phosphorylation is elevated during mitosis in mih1∆ cells, Cdk1 is dephosphorylated as cells progress into G₁, suggesting that additional phosphatases regulate Cdk1 dephosphorylation. Here we show that the phosphatase Ptp1 also regulates Cdk1 dephosphorylation in vivo and can directly dephosphorylate Cdk1 in vitro. Using a novel in vivo phosphatase assay, we also show that PP2A bound to Rts1, the budding yeast B56-regulatory subunit, regulates dephosphorylation of Cdk1 independently of a function regulating Swe1, Mih1, or Ptp1, suggesting that PP2A^Rts1 either directly dephosphorylates Cdk1-Y19 or regulates an unidentified phosphatase.