Publications by authors named "Kenneth H Moberg"

37 Publications

A protocol to detect neurodegeneration in whole-brain mounts using advanced microscopy.

STAR Protoc 2021 Sep 28;2(3):100689. Epub 2021 Jul 28.

Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA.

is an excellent model organism to study neurodegeneration. Assessing evident neurodegeneration within the fly brain involves the laborious preparation of thin-sectioned H&E-stained heads to visualize brain vacuole degeneration. Here, we present an advanced microscopy-based protocol, without the need for sectioning, to detect vacuole degeneration within whole fly brains by applying commonly used stains to reveal the brain parenchyma. This approach preserves the whole-brain architecture and enables rapid, reproducible, and quantitative analyses of vacuole-like degeneration associated with specific brain regions. For complete details on the use and execution of this protocol, please refer to Behnke et al. (2021).
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http://dx.doi.org/10.1016/j.xpro.2021.100689DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8339312PMC
September 2021

The RNA-binding protein Nab2 regulates the proteome of the developing Drosophila brain.

J Biol Chem 2021 07 15;297(1):100877. Epub 2021 Jun 15.

Department of Cell Biology, Emory University School of Medicine, Emory University, Atlanta, Georgia, USA. Electronic address:

The human ZC3H14 gene, which encodes a ubiquitously expressed polyadenosine zinc finger RNA-binding protein, is mutated in an inherited form of autosomal recessive, nonsyndromic intellectual disability. To gain insight into neurological functions of ZC3H14, we previously developed a Drosophila melanogaster model of ZC3H14 loss by deleting the fly ortholog, Nab2. Studies in this invertebrate model revealed that Nab2 controls final patterns of neuron projection within fully developed adult brains, but the role of Nab2 during development of the Drosophila brain is not known. Here, we identify roles for Nab2 in controlling the dynamic growth of axons in the developing brain mushroom bodies, which support olfactory learning and memory, and regulating abundance of a small fraction of the total brain proteome. The group of Nab2-regulated brain proteins, identified by quantitative proteomic analysis, includes the microtubule-binding protein Futsch, the neuronal Ig-family transmembrane protein turtle, the glial:neuron adhesion protein contactin, the Rac GTPase-activating protein tumbleweed, and the planar cell polarity factor Van Gogh, which collectively link Nab2 to the processes of brain morphogenesis, neuroblast proliferation, circadian sleep/wake cycles, and synaptic development. Overall, these data indicate that Nab2 controls the abundance of a subset of brain proteins during the active process of wiring the pupal brain mushroom body and thus provide a window into potentially conserved functions of the Nab2/ZC3H14 RNA-binding proteins in neurodevelopment.
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http://dx.doi.org/10.1016/j.jbc.2021.100877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8260979PMC
July 2021

Repetitive mild head trauma induces activity mediated lifelong brain deficits in a novel Drosophila model.

Sci Rep 2021 May 6;11(1):9738. Epub 2021 May 6.

Department of Cell Biology, Emory University School of Medicine, Atlanta, GA, 30322, USA.

Mild head trauma, including concussion, can lead to chronic brain dysfunction and degeneration but the underlying mechanisms remain poorly understood. Here, we developed a novel head impact system to investigate the long-term effects of mild head trauma on brain structure and function, as well as the underlying mechanisms in Drosophila melanogaster. We find that Drosophila subjected to repetitive head impacts develop long-term deficits, including impaired startle-induced climbing, progressive brain degeneration, and shortened lifespan, all of which are substantially exacerbated in female flies. Interestingly, head impacts elicit an elevation in neuronal activity and its acute suppression abrogates the detrimental effects in female flies. Together, our findings validate Drosophila as a suitable model system for investigating the long-term effects of mild head trauma, suggest an increased vulnerability to brain injury in female flies, and indicate that early altered neuronal excitability may be a key mechanism linking mild brain trauma to chronic degeneration.
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http://dx.doi.org/10.1038/s41598-021-89121-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8102574PMC
May 2021

A Genetic Screen Links the Disease-Associated Nab2 RNA-Binding Protein to the Planar Cell Polarity Pathway in .

G3 (Bethesda) 2020 10 5;10(10):3575-3583. Epub 2020 Oct 5.

Emory University School of Medicine, Department of Cell Biology, Atlanta, GA 30322

Mutations in the gene encoding the ubiquitously expressed RNA-binding protein ZC3H14 result in a non-syndromic form of autosomal recessive intellectual disability in humans. Studies in have defined roles for the ZC3H14 ortholog, Nab2 (aka Nab2 or dNab2), in axon guidance and memory due in part to interaction with a second RNA-binding protein, the fly Fragile X homolog Fmr1, and coregulation of shared Nab2-Fmr1 target mRNAs. Despite these advances, neurodevelopmental mechanisms that underlie defective axonogenesis in mutants remain undefined. null phenotypes in the brain mushroom bodies (MBs) resemble defects caused by alleles that disrupt the planar cell polarity (PCP) pathway, which regulates planar orientation of static and motile cells via a non-canonical arm of the Wnt/Wg pathway. A kinked bristle phenotype in surviving mutant adults additionally suggests a defect in F-actin polymerization and bundling, a PCP-regulated processes. To test for Nab2-PCP genetic interactions, a collection of PCP mutant alleles was screened for modification of a rough-eye phenotype produced by Nab2 overexpression in the eye ( ) and, subsequently, for modification of a viability defect among nulls. Multiple PCP alleles dominantly modify eye roughening and a subset rescue low survival and thoracic bristle kinking in zygotic nulls. Collectively, these genetic interactions identify the PCP pathway as a potential target of the Nab2 RNA-binding protein in developing eye and wing tissues and suggest that altered PCP signaling could contribute to neurological defects that result from loss of Nab2 or its vertebrate ortholog ZC3H14.
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http://dx.doi.org/10.1534/g3.120.401637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7534439PMC
October 2020

A Drosophila model of Pontocerebellar Hypoplasia reveals a critical role for the RNA exosome in neurons.

PLoS Genet 2020 07 9;16(7):e1008901. Epub 2020 Jul 9.

Department of Biology, RRC 1021, Emory University, NE, Atlanta, Georgia, United States of America.

The RNA exosome is an evolutionarily-conserved ribonuclease complex critically important for precise processing and/or complete degradation of a variety of cellular RNAs. The recent discovery that mutations in genes encoding structural RNA exosome subunits cause tissue-specific diseases makes defining the role of this complex within specific tissues critically important. Mutations in the RNA exosome component 3 (EXOSC3) gene cause Pontocerebellar Hypoplasia Type 1b (PCH1b), an autosomal recessive neurologic disorder. The majority of disease-linked mutations are missense mutations that alter evolutionarily-conserved regions of EXOSC3. The tissue-specific defects caused by these amino acid changes in EXOSC3 are challenging to understand based on current models of RNA exosome function with only limited analysis of the complex in any multicellular model in vivo. The goal of this study is to provide insight into how mutations in EXOSC3 impact the function of the RNA exosome. To assess the tissue-specific roles and requirements for the Drosophila ortholog of EXOSC3 termed Rrp40, we utilized tissue-specific RNAi drivers. Depletion of Rrp40 in different tissues reveals a general requirement for Rrp40 in the development of many tissues including the brain, but also highlight an age-dependent requirement for Rrp40 in neurons. To assess the functional consequences of the specific amino acid substitutions in EXOSC3 that cause PCH1b, we used CRISPR/Cas9 gene editing technology to generate flies that model this RNA exosome-linked disease. These flies show reduced viability; however, the surviving animals exhibit a spectrum of behavioral and morphological phenotypes. RNA-seq analysis of these Drosophila Rrp40 mutants reveals increases in the steady-state levels of specific mRNAs and ncRNAs, some of which are central to neuronal function. In particular, Arc1 mRNA, which encodes a key regulator of synaptic plasticity, is increased in the Drosophila Rrp40 mutants. Taken together, this study defines a requirement for the RNA exosome in specific tissues/cell types and provides insight into how defects in RNA exosome function caused by specific amino acid substitutions that occur in PCH1b can contribute to neuronal dysfunction.
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http://dx.doi.org/10.1371/journal.pgen.1008901DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7373318PMC
July 2020

Enhancer architecture sensitizes cell specific responses to gene dose via a bind and discard mechanism.

Elife 2020 04 16;9. Epub 2020 Apr 16.

Division of Developmental Biology, Cincinnati Children's Hospital, Cincinnati, United States.

Notch pathway haploinsufficiency can cause severe developmental syndromes with highly variable penetrance. Currently, we have a limited mechanistic understanding of phenotype variability due to gene dosage. Here, we unexpectedly found that inserting an enhancer containing pioneer transcription factor sites coupled to Notch dimer sites can induce a subset of haploinsufficiency phenotypes in with wild type gene dose. Using genetics, we show that this enhancer induces Notch phenotypes in a Cdk8-dependent, transcription-independent manner. We further combined mathematical modeling with quantitative trait and expression analysis to build a model that describes how changes in Notch signal production versus degradation differentially impact cellular outcomes that require long versus short signal duration. Altogether, these findings support a 'bind and discard' mechanism in which enhancers with specific binding sites promote rapid Cdk8-dependent Notch turnover, and thereby reduce Notch-dependent transcription at other loci and sensitize tissues to gene dose based upon signal duration.
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http://dx.doi.org/10.7554/eLife.53659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7213981PMC
April 2020

Rare variants in MYH15 modify amyotrophic lateral sclerosis risk.

Hum Mol Genet 2019 07;28(14):2309-2318

Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA.

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by progressive muscular atrophy and respiratory failure. The G4C2 repeat expansion in the C9orf72 gene is the most prevalent genetic risk for ALS. Mutation carriers (C9ALS) display variability in phenotypes such as age-at-onset and duration, suggesting the existence of additional genetic factors. Here we introduce a three-step gene discovery strategy to identify genetic factors modifying the risk of both C9ALS and sporadic ALS (sALS) using limited samples. We first identified 135 candidate genetic modifiers of C9ALS using whole-genome sequencing (WGS) of extreme C9ALS cases diagnosed ~30 years apart. We then performed an unbiased genetic screen using a Drosophila model of the G4C2 repeat expansion with the genes identified from WGS analysis. This genetic screen identified the novel genetic interaction between G4C2 repeat-associated toxicity and 18 genetic factors, suggesting their potential association with C9ALS risk. We went on to test if 14 out of the 18 genes, those which were not known to be risk factors for ALS previously, are also associated with ALS risk in sALS cases. Gene-based-statistical analyses of targeted resequencing and WGS were performed. These analyses together reveal that rare variants in MYH15 represent a likely genetic risk factor for ALS. Furthermore, we show that MYH15 could modulate the toxicity of dipeptides produced from expanded G4C2 repeat. Our study presented here demonstrates the power of combining WGS with fly genetics to facilitate the discovery of fundamental genetic components of complex traits with a limited number of samples.
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http://dx.doi.org/10.1093/hmg/ddz063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6606848PMC
July 2019

Active N-Methyladenine Demethylation by DMAD Regulates Gene Expression by Coordinating with Polycomb Protein in Neurons.

Mol Cell 2018 09 2;71(5):848-857.e6. Epub 2018 Aug 2.

Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322, USA. Electronic address:

A ten-eleven translocation (TET) ortholog exists as a DNA N-methyladenine (6mA) demethylase (DMAD) in Drosophila. However, the molecular roles of 6mA and DMAD remain unexplored. Through genome-wide 6mA and transcriptome profiling in Drosophila brains and neuronal cells, we found that 6mA may epigenetically regulate a group of genes involved in neurodevelopment and neuronal functions. Mechanistically, DMAD interacts with the Trithorax-related complex protein Wds to maintain active transcription by dynamically demethylating intragenic 6mA. Accumulation of 6mA by depleting DMAD coordinates with Polycomb proteins and contributes to transcriptional repression of these genes. Our findings suggest that active 6mA demethylation by DMAD plays essential roles in fly CNS by orchestrating through added epigenetic mechanisms.
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http://dx.doi.org/10.1016/j.molcel.2018.07.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6136845PMC
September 2018

The SWI2/SNF2 Gene Product Represses Cell Death in .

G3 (Bethesda) 2018 07 2;8(7):2355-2360. Epub 2018 Jul 2.

Biology Department, Emory University, Atlanta, Georgia 30322

The Drosophila locus encodes DNA-dependent ATPases of the SWI2/SNF2 class. This class of chromatin remodeler is associated with an array of cellular activities encompassing transcription, replication, repair and recombination. Moreover, was observed initially to maintain a repressive chromatin state via genetic interaction studies with homeotic genes. Although mutations were also characterized with a cell death phenotype, its association with a death pathway has not been investigated. Here we have used targeted RNA interference to depress function in the wing. Resultant wing damage phenotypes were found to be enhanced through overexpression of pro-apoptotic loci, and suppressed through loss of function of these loci. Loss of wing margin and blade tissue was correlated with activation of the effector Caspase Dcp-1, a marker for apoptosis. The affected wing regions also exhibited lower levels of the DIAP1 protein, an inhibitor of apoptosis. The lower level of DIAP1 protein was not correlated with an effect on the activity of a DIAP1 gene transgenic reporter (), suggesting that loss of DIAP1 occurred post transcriptionally. In some cases excessive cell proliferation within the targeted tissue, measured through BrdU incorporation, was also observed. Finally, we used a transgenic reporter construct to monitor the chromatin state upstream of the proapoptotic locus. In genotypes exhibiting targeted loss and wing phenotypes, we observed increased reporter activity only in the affected areas. These data support the conclusion that normally functions to maintain pro-apoptotic genes in a repressed state.
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http://dx.doi.org/10.1534/g3.118.200228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6027882PMC
July 2018

The Conserved, Disease-Associated RNA Binding Protein dNab2 Interacts with the Fragile X Protein Ortholog in Drosophila Neurons.

Cell Rep 2017 08;20(6):1372-1384

Department of Cell Biology, Emory University and Emory University School of Medicine, Atlanta, GA 30322, USA. Electronic address:

The Drosophila dNab2 protein is an ortholog of human ZC3H14, a poly(A) RNA binding protein required for intellectual function. dNab2 supports memory and axon projection, but its molecular role in neurons is undefined. Here, we present a network of interactions that links dNab2 to cytoplasmic control of neuronal mRNAs in conjunction with the fragile X protein ortholog dFMRP. dNab2 and dfmr1 interact genetically in control of neurodevelopment and olfactory memory, and their encoded proteins co-localize in puncta within neuronal processes. dNab2 regulates CaMKII, but not futsch, implying a selective role in control of dFMRP-bound transcripts. Reciprocally, dFMRP and vertebrate FMRP restrict mRNA poly(A) tail length, similar to dNab2/ZC3H14. Parallel studies of murine hippocampal neurons indicate that ZC3H14 is also a cytoplasmic regulator of neuronal mRNAs. Altogether, these findings suggest that dNab2 represses expression of a subset of dFMRP-target mRNAs, which could underlie brain-specific defects in patients lacking ZC3H14.
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http://dx.doi.org/10.1016/j.celrep.2017.07.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5577809PMC
August 2017

The RNA-binding protein, ZC3H14, is required for proper poly(A) tail length control, expression of synaptic proteins, and brain function in mice.

Hum Mol Genet 2017 10;26(19):3663-3681

Department of Biology.

A number of mutations in genes that encode ubiquitously expressed RNA-binding proteins cause tissue specific disease. Many of these diseases are neurological in nature revealing critical roles for this class of proteins in the brain. We recently identified mutations in a gene that encodes a ubiquitously expressed polyadenosine RNA-binding protein, ZC3H14 (Zinc finger CysCysCysHis domain-containing protein 14), that cause a nonsyndromic, autosomal recessive form of intellectual disability. This finding reveals the molecular basis for disease and provides evidence that ZC3H14 is essential for proper brain function. To investigate the role of ZC3H14 in the mammalian brain, we generated a mouse in which the first common exon of the ZC3H14 gene, exon 13 is removed (Zc3h14Δex13/Δex13) leading to a truncated ZC3H14 protein. We report here that, as in the patients, Zc3h14 is not essential in mice. Utilizing these Zc3h14Δex13/Δex13mice, we provide the first in vivo functional characterization of ZC3H14 as a regulator of RNA poly(A) tail length. The Zc3h14Δex13/Δex13 mice show enlarged lateral ventricles in the brain as well as impaired working memory. Proteomic analysis comparing the hippocampi of Zc3h14+/+ and Zc3h14Δex13/Δex13 mice reveals dysregulation of several pathways that are important for proper brain function and thus sheds light onto which pathways are most affected by the loss of ZC3H14. Among the proteins increased in the hippocampi of Zc3h14Δex13/Δex13 mice compared to control are key synaptic proteins including CaMK2a. This newly generated mouse serves as a tool to study the function of ZC3H14 in vivo.
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http://dx.doi.org/10.1093/hmg/ddx248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5886104PMC
October 2017

The ecdysone receptor coactivator Taiman links Yorkie to transcriptional control of germline stem cell factors in somatic tissue.

Dev Cell 2015 Jul 2;34(2):168-80. Epub 2015 Jul 2.

Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA. Electronic address:

The Hippo pathway is a conserved signaling cascade that modulates tissue growth. Although its core elements are well defined, factors modulating Hippo transcriptional outputs remain elusive. Here we show that components of the steroid-responsive ecdysone (Ec) pathway modulate Hippo transcriptional effects in imaginal disc cells. The Ec receptor coactivator Taiman (Tai) interacts with the Hippo transcriptional coactivator Yorkie (Yki) and promotes expression of canonical Yki-responsive genes. Tai enhances Yki-driven growth, while Tai loss, or a form of Tai unable to bind Yki, suppresses Yki-driven tissue growth. This growth suppression is not correlated with impaired induction of canonical Hippo-responsive genes but with suppression of a distinct pro-growth program of Yki-induced/Tai-dependent genes, including the germline stem cell factors nanos and piwi. These data reveal Hippo/Ec pathway crosstalk in the form a Yki-Tai complex that collaboratively induces germline genes as part of a transcriptional program that is normally repressed in developing somatic epithelia.
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http://dx.doi.org/10.1016/j.devcel.2015.05.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4519380PMC
July 2015

The Drosophila ortholog of the Zc3h14 RNA binding protein acts within neurons to pattern axon projection in the developing brain.

Dev Neurobiol 2016 Jan 1;76(1):93-106. Epub 2015 Jun 1.

Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia, 30322.

The dNab2 polyadenosine RNA binding protein is the D. melanogaster ortholog of the vertebrate ZC3H14 protein, which is lost in a form of inherited intellectual disability (ID). Human ZC3H14 can rescue D. melanogaster dNab2 mutant phenotypes when expressed in all neurons of the developing nervous system, suggesting that dNab2/ZC3H14 performs well-conserved roles in neurons. However, the cellular and molecular requirements for dNab2/ZC3H14 in the developing nervous system have not been defined in any organism. Here we show that dNab2 is autonomously required within neurons to pattern axon projection from Kenyon neurons into the mushroom bodies, which are required for associative olfactory learning and memory in insects. Mushroom body axons lacking dNab2 project aberrantly across the brain midline and also show evidence of defective branching. Coupled with the prior finding that ZC3H14 is highly expressed in rodent hippocampal neurons, this requirement for dNab2 in mushroom body neurons suggests that dNab2/ZC3H14 has a conserved role in supporting axon projection and branching. Consistent with this idea, loss of dNab2 impairs short-term memory in a courtship conditioning assay. Taken together these results reveal a cell-autonomous requirement for the dNab2 RNA binding protein in mushroom body development and provide a window into potential neurodevelopmental functions of the human ZC3H14 protein.
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http://dx.doi.org/10.1002/dneu.22301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4644733PMC
January 2016

Increased expression of the PI3K enhancer PIKE mediates deficits in synaptic plasticity and behavior in fragile X syndrome.

Cell Rep 2015 May 23;11(5):727-36. Epub 2015 Apr 23.

Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA. Electronic address:

The PI3K enhancer PIKE links PI3K catalytic subunits to group 1 metabotropic glutamate receptors (mGlu1/5) and activates PI3K signaling. The roles of PIKE in synaptic plasticity and the etiology of mental disorders are unknown. Here, we show that increased PIKE expression is a key mediator of impaired mGlu1/5-dependent neuronal plasticity in mouse and fly models of the inherited intellectual disability fragile X syndrome (FXS). Normalizing elevated PIKE protein levels in FXS mice reversed deficits in molecular and cellular plasticity and improved behavior. Notably, PIKE reduction rescued PI3K-dependent and -independent neuronal defects in FXS. We further show that PI3K signaling is increased in a fly model of FXS and that genetic reduction of the Drosophila ortholog of PIKE, CenG1A rescued excessive PI3K signaling, mushroom body defects, and impaired short-term memory in these flies. Our results demonstrate a crucial role of increased PIKE expression in exaggerated mGlu1/5 signaling causing neuronal defects in FXS.
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http://dx.doi.org/10.1016/j.celrep.2015.03.060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4418204PMC
May 2015

A conserved role for the zinc finger polyadenosine RNA binding protein, ZC3H14, in control of poly(A) tail length.

RNA 2014 May 26;20(5):681-8. Epub 2014 Mar 26.

The ZC3H14 gene, which encodes a ubiquitously expressed, evolutionarily conserved, nuclear, zinc finger polyadenosine RNA-binding protein, was recently linked to autosomal recessive, nonsyndromic intellectual disability. Although studies have been carried out to examine the function of putative orthologs of ZC3H14 in Saccharomyces cerevisiae, where the protein is termed Nab2, and Drosophila, where the protein has been designated dNab2, little is known about the function of mammalian ZC3H14. Work from both budding yeast and flies implicates Nab2/dNab2 in poly(A) tail length control, while a role in poly(A) RNA export from the nucleus has been reported only for budding yeast. Here we provide the first functional characterization of ZC3H14. Analysis of ZC3H14 function in a neuronal cell line as well as in vivo complementation studies in a Drosophila model identify a role for ZC3H14 in proper control of poly(A) tail length in neuronal cells. Furthermore, we show here that human ZC3H14 can functionally substitute for dNab2 in fly neurons and can rescue defects in development and locomotion that are present in dNab2 null flies. These rescue experiments provide evidence that this zinc finger-containing class of nuclear polyadenosine RNA-binding proteins plays an evolutionarily conserved role in controlling the length of the poly(A) tail in neurons.
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http://dx.doi.org/10.1261/rna.043984.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3988569PMC
May 2014

The archipelago ubiquitin ligase subunit acts in target tissue to restrict tracheal terminal cell branching and hypoxic-induced gene expression.

PLoS Genet 2013 14;9(2):e1003314. Epub 2013 Feb 14.

Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia, USA.

The Drosophila melanogaster gene archipelago (ago) encodes the F-box/WD-repeat protein substrate specificity factor for an SCF (Skp/Cullin/F-box)-type polyubiquitin ligase that inhibits tumor-like growth by targeting proteins for degradation by the proteasome. The Ago protein is expressed widely in the fly embryo and larva and promotes degradation of pro-proliferative proteins in mitotically active cells. However the requirement for Ago in post-mitotic developmental processes remains largely unexplored. Here we show that Ago is an antagonist of the physiologic response to low oxygen (hypoxia). Reducing Ago activity in larval muscle cells elicits enhanced branching of nearby tracheal terminal cells in normoxia. This tracheogenic phenotype shows a genetic dependence on sima, which encodes the HIF-1α subunit of the hypoxia-inducible transcription factor dHIF and its target the FGF ligand branchless (bnl), and is enhanced by depletion of the Drosophila Von Hippel Lindau (dVHL) factor, which is a subunit of an oxygen-dependent ubiquitin ligase that degrades Sima/HIF-1α protein in metazoan cells. Genetic reduction of ago results in constitutive expression of some hypoxia-inducible genes in normoxia, increases the sensitivity of others to mild hypoxic stimulus, and enhances the ability of adult flies to recover from hypoxic stupor. As a molecular correlate to these genetic data, we find that Ago physically associates with Sima and restricts Sima levels in vivo. Collectively, these findings identify Ago as a required element of a circuit that suppresses the tracheogenic activity of larval muscle cells by antagonizing the Sima-mediated transcriptional response to hypoxia.
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http://dx.doi.org/10.1371/journal.pgen.1003314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3573119PMC
June 2013

Stable isotope labeling with amino acids in Drosophila for quantifying proteins and modifications.

J Proteome Res 2012 Sep 10;11(9):4403-12. Epub 2012 Aug 10.

Department of Human Genetics, Center for Neurodegenerative Diseases, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

Drosophila melanogaster is a common animal model for genetics studies, and quantitative proteomics studies of the fly are emerging. Here, we present in detail the development of a procedure to incorporate stable isotope-labeled amino acids into the fly proteome. In the method of stable isotope labeling with amino acids in Drosophila melanogaster (SILAC fly), flies were fed with SILAC-labeled yeast grown with modified media, enabling near complete labeling in a single generation. Biological variation in the proteome among individual flies was evaluated in a series of null experiments. We further applied the SILAC fly method to profile proteins from a model of fragile X syndrome, the most common cause of inherited mental retardation in human. The analysis identified a number of altered proteins in the disease model, including actin-binding protein profilin and microtubulin-associated protein futsch. The change of both proteins was validated by immunoblotting analysis. Moreover, we extended the SILAC fly strategy to study the dynamics of protein ubiquitination during the fly life span (from day 1 to day 30), by measuring the level of ubiquitin along with two major polyubiquitin chains (K48 and K63 linkages). The results show that the abundance of protein ubiquitination and the two major linkages do not change significantly within the measured age range. Together, the data demonstrate the application of the SILAC principle in D. melanogaster, facilitating the integration of powerful fly genomics with emerging proteomics.
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http://dx.doi.org/10.1021/pr300613cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3443408PMC
September 2012

Regulation of Yorkie activity in Drosophila imaginal discs by the Hedgehog receptor gene patched.

Mech Dev 2012 Sep-Dec;129(9-12):339-49. Epub 2012 Jun 15.

Department of Biology, University of Detroit Mercy, Detroit, MI, USA.

The Hedgehog (Hh) pathway was first defined by its role in segment polarity in the Drosophila melanogaster embryonic epidermis and has since been linked to many aspects of vertebrate development and disease. In humans, mutation of the Patched1 (PTCH1) gene, which encodes an inhibitor of Hh signaling, leads to tumors of the skin and pediatric brain. Despite the high level of conservation between the vertebrate and invertebrate Hh pathways, studies in Drosophila have yet to find direct evidence that ptc limits organ size. Here we report identification of Drosophila ptc in a screen for mutations that require a synergistic apoptotic block in order to drive overgrowth. Developing imaginal discs containing clones of ptc mutant cells immortalized by the concurrent loss of the Apaf-1-related killer (Ark) gene are overgrown due, in large part, to the overgrowth of wild type portions of these discs. This phenotype correlates with overexpression of the morphogen Dpp in ptc,Ark double-mutant cells, leading to elevated phosphorylation of the Dpp pathway effector Mad (p-Mad) in cells surrounding ptc,Ark mutant clones. p-Mad functions with the Hippo pathway oncoprotein Yorkie (Yki) to induce expression of the pro-growth/anti-apoptotic microRNA bantam. Accordingly, Yki activity is elevated among wild type cells surrounding ptc,Ark clones and alleles of bantam and yki dominantly suppress the enlarged-disc phenotype produced by loss of ptc. These data suggest that ptc can regulate Yki in a non-cell autonomous manner and reveal an intercellular link between the Hh and Hippo pathways that may contribute to growth-regulatory properties of the Hh pathway in development and disease.
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http://dx.doi.org/10.1016/j.mod.2012.05.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3668547PMC
June 2013

Drosophila endocytic neoplastic tumor suppressor genes regulate Sav/Wts/Hpo signaling and the c-Jun N-terminal kinase pathway.

Cell Cycle 2011 Dec 1;10(23):4110-8. Epub 2011 Dec 1.

Department of Cell Biology, Emory University School of Medicine, Atlanta, GA, USA.

Genetic screens in the fruit fly Drosophila melanogaster have identified a class of neoplastic tumor suppressor genes (endocytic nTSGs), which encode proteins that localize to endosomes and facilitate the trafficking of membrane-bound receptors and adhesion molecules into the degradative lysosome. Loss of endocytic nTSGs transforms imaginal disc epithelia into highly proliferative, invasive tissues that fail to differentiate and display defects in cellular apicobasal polarity, adhesion and tissue architecture. As vertebrate homologs of some Drosophila nTSGs are linked to tumor formation, identifying molecular changes in signaling associated with nTSG loss could inform understanding of neoplastic transformation in vertebrates. Here we show that mutations in genes that act at multiple steps of the endolysosomal pathway lead to autonomous activation of the Sav/Wts/Hpo (SWH) transcriptional effector Yki (YAP/TAZ in vertebrates) and the Jun N-terminal kinase (JNK), which is known to promote Yki activity in cells with disrupted polarity. Yki and JNK activity are elevated by mutations at multiple steps in the endolysosomal pathway including mutations in the AP-2σ gene, which encodes a component of the AP-2 adaptor complex that recruits cargoes into clathrin-coated pits for subsequent internalization. Moreover, reduction of JNK activity can decrease elevated Yki-signaling caused by altered endocytosis. These studies reveal a broad requirement for components of the endocytic pathway in regulating SWH and JNK outputs, and place Drosophila endocytic nTSGs into a network that involving two major signaling pathways implicated in oncogenesis.
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http://dx.doi.org/10.4161/cc.10.23.18243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3272291PMC
December 2011

Cell-cell junctions: α-catenin and E-cadherin help fence in Yap1.

Curr Biol 2011 Nov;21(21):R890-2

Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA.

Metazoan cells translate adhesive events with neighbors into anti-proliferative signals in the nucleus. The cadherin-catenin adhesion complex has long been suspected of playing a key role in this process, and three recent papers suggest that it does so by modulating subcellular localization of the Hippo pathway component Yap1.
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http://dx.doi.org/10.1016/j.cub.2011.09.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5695222PMC
November 2011

Aeromonas salmonicida-secreted protein AopP is a potent inducer of apoptosis in a mammalian and a Drosophila model.

Cell Microbiol 2012 Feb 21;14(2):274-85. Epub 2011 Nov 21.

Departments of Pathology and Laboratory Medicine Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA.

Some pathogens are able to establish themselves within the host because they have evolved mechanisms to disrupt host innate immunity. For example, a number of pathogens secrete preformed effector proteins via type III secretion apparatuses that influence innate immune or apoptotic signalling pathways. One group of effector proteins that usurp innate immune signalling is the YopJ-like family of bacterial effector proteins, which includes AopP from Aeromonas salmonicida. Aeromonas species are known to cause gastrointestinal disease in humans, and are associated mainly with subcutaneous wound infections and septicaemia in other metazoans, particularly fish. AopP has been reported to have inhibitory activity against the NF-κB pathway in cultured cells, although the pathological outcomes of AopP activity have not been examined. Here, we show that AopP has potent pro-apoptotic activity when expressed in cultured mammalian macrophage or epithelial cells, or when ectopically expressed in Drosophila melanogaster haemocytes or imaginal disk epithelial cells. Furthermore, apoptosis was significantly elevated upon concurrent AopP expression and TNF-α cellular stimulation. Together, our results demonstrate how the specificity of a YopJ-like protein towards signalling pathways directly governs cellular pathological outcome in disease.
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http://dx.doi.org/10.1111/j.1462-5822.2011.01717.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3267377PMC
February 2012

Mutation of the conserved polyadenosine RNA binding protein, ZC3H14/dNab2, impairs neural function in Drosophila and humans.

Proc Natl Acad Sci U S A 2011 Jul 6;108(30):12390-5. Epub 2011 Jul 6.

Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA.

Here we report a human intellectual disability disease locus on chromosome 14q31.3 corresponding to mutation of the ZC3H14 gene that encodes a conserved polyadenosine RNA binding protein. We identify ZC3H14 mRNA transcripts in the human central nervous system, and we find that rodent ZC3H14 protein is expressed in hippocampal neurons and colocalizes with poly(A) RNA in neuronal cell bodies. A Drosophila melanogaster model of this disease created by mutation of the gene encoding the ZC3H14 ortholog dNab2, which also binds polyadenosine RNA, reveals that dNab2 is essential for development and required in neurons for normal locomotion and flight. Biochemical and genetic data indicate that dNab2 restricts bulk poly(A) tail length in vivo, suggesting that this function may underlie its role in development and disease. These studies reveal a conserved requirement for ZC3H14/dNab2 in the metazoan nervous system and identify a poly(A) RNA binding protein associated with a human brain disorder.
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http://dx.doi.org/10.1073/pnas.1107103108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3145741PMC
July 2011

A screen for conditional growth suppressor genes identifies the Drosophila homolog of HD-PTP as a regulator of the oncoprotein Yorkie.

Dev Cell 2011 May;20(5):700-12

Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA.

Mammalian cancers depend on "multiple hits," some of which promote growth and some of which block apoptosis. We screened for mutations that require a synergistic block in apoptosis to promote tissue overgrowth and identified myopic (mop), the Drosophila homolog of the candidate tumor-suppressor and endosomal regulator His-domain protein tyrosine phosphatase (HD-PTP). We find that Myopic regulates the Salvador/Warts/Hippo (SWH) tumor suppressor pathway: Myopic PPxY motifs bind conserved residues in the WW domains of the transcriptional coactivator Yorkie, and Myopic colocalizes with Yorkie at endosomes. Myopic controls Yorkie endosomal association and protein levels, ultimately influencing expression of some Yorkie target genes. However, the antiapoptotic gene diap1 is not affected, which may explain the conditional nature of the myopic growth phenotype. These data establish Myopic as a Yorkie regulator and implicate Myopic-dependent association of Yorkie with endosomal compartments as a regulatory step in nuclear outputs of the SWH pathway.
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http://dx.doi.org/10.1016/j.devcel.2011.04.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3386645PMC
May 2011

Notch-dependent expression of the archipelago ubiquitin ligase subunit in the Drosophila eye.

Development 2011 Jan 9;138(2):251-60. Epub 2010 Dec 9.

Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA.

archipelago (ago)/Fbw7 encodes a conserved protein that functions as the substrate-receptor component of a polyubiquitin ligase that suppresses tissue growth in flies and tumorigenesis in vertebrates. Ago/Fbw7 targets multiple proteins for degradation, including the G1-S regulator Cyclin E and the oncoprotein dMyc/c-Myc. Despite prominent roles in growth control, little is known about the signals that regulate Ago/Fbw7 abundance in developing tissues. Here we use the Drosophila eye as a model to identify developmental signals that regulate ago expression. We find that expression of ago mRNA and protein is induced by passage of the morphogenetic furrow (MF) and identify the hedgehog (hh) and Notch (N) pathways as elements of this inductive mechanism. Cells mutant for N pathway components, or hh-defective cells that express reduced levels of the Notch ligand Delta, fail to upregulate ago transcription in the region of the MF; reciprocally, ectopic N activation in eye discs induces expression of ago mRNA. A fragment of the ago promoter that contains consensus binding sites for the N pathway transcription factor Su(H) is bound by Su(H) and confers N-inducibility in cultured cells. The failure to upregulate ago in N pathway mutant cells correlates with accumulation of the SCF-Ago target Cyclin E in the area of the MF, and this is rescued by re-expression of ago. These data suggest a model in which N acts through ago to restrict levels of the pro-mitotic factor Cyclin E. This N-Ago-Cyclin E link represents a significant new cell cycle regulatory mechanism in the developing eye.
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http://dx.doi.org/10.1242/dev.054429DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3005602PMC
January 2011

A Drosophila melanogaster model of classic galactosemia.

Dis Model Mech 2010 Sep-Oct;3(9-10):618-27. Epub 2010 Jun 2.

Graduate Program in Biochemistry, Cell and Developmental Biology, Emory University, Atlanta, GA 30322, USA.

Classic galactosemia is a potentially lethal disorder that results from profound impairment of galactose-1-phosphate uridylyltransferase (GALT). Despite decades of research, the underlying pathophysiology of classic galactosemia remains unclear, in part owing to the lack of an appropriate animal model. Here, we report the establishment of a Drosophila melanogaster model of classic galactosemia; this is the first whole-animal genetic model to mimic aspects of the patient phenotype. Analogous to humans, GALT-deficient D. melanogaster survive under conditions of galactose restriction, but accumulate elevated levels of galactose-1-phosphate and succumb during larval development following galactose exposure. As in patients, the potentially lethal damage is reversible if dietary galactose restriction is initiated early in life. GALT-deficient Drosophila also exhibit locomotor complications despite dietary galactose restriction, and both the acute and long-term complications can be rescued by transgenic expression of human GALT. Using this new Drosophila model, we have begun to dissect the timing, extent and mechanism(s) of galactose sensitivity in the absence of GALT activity.
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http://dx.doi.org/10.1242/dmm.005041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2931538PMC
December 2010

UDP-galactose 4' epimerase (GALE) is essential for development of Drosophila melanogaster.

Dis Model Mech 2010 Sep-Oct;3(9-10):628-38. Epub 2010 Jun 2.

Graduate Program in Biochemistry, Cell and Developmental Biology, Emory University, Atlanta, GA 30322, USA.

UDP-galactose 4' epimerase (GALE) catalyzes the interconversion of UDP-galactose and UDP-glucose in the final step of the Leloir pathway; human GALE (hGALE) also interconverts UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine. GALE therefore plays key roles in the metabolism of dietary galactose, in the production of endogenous galactose, and in maintaining the ratios of key substrates for glycoprotein and glycolipid biosynthesis. Partial impairment of hGALE results in the potentially lethal disorder epimerase-deficiency galactosemia. We report here the generation and initial characterization of a first whole-animal model of GALE deficiency using the fruit fly Drosophila melanogaster. Our results confirm that GALE function is essential in developing animals; Drosophila lacking GALE die as embryos but are rescued by the expression of a human GALE transgene. Larvae in which GALE has been conditionally knocked down die within days of GALE loss. Conditional knockdown and transgene expression studies further demonstrate that GALE expression in the gut primordium and Malpighian tubules is both necessary and sufficient for survival. Finally, like patients with generalized epimerase deficiency galactosemia, Drosophila with partial GALE loss survive in the absence of galactose but succumb in development if exposed to dietary galactose. These data establish the utility of the fly model of GALE deficiency and set the stage for future studies to define the mechanism(s) and modifiers of outcome in epimerase deficiency galactosemia.
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http://dx.doi.org/10.1242/dmm.005058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2931539PMC
December 2010

Crumbs regulates Salvador/Warts/Hippo signaling in Drosophila via the FERM-domain protein Expanded.

Curr Biol 2010 Apr 1;20(7):582-90. Epub 2010 Apr 1.

Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA.

Background: Altered expression of apicobasal polarity factors is associated with cancer in vertebrates and tissue overgrowth in invertebrates, yet the mechanisms by which these factors affect growth-regulatory pathways are not well defined. We have tested the basis of an overgrowth phenotype driven by the Drosophila protein Crumbs (Crb), which nucleates an apical membrane complex that functionally interacts with the Par6/Par3/aPKC and Scrib/Dlg/Lgl apicobasal polarity complexes.

Results: We find that Crb-driven growth is dependent upon the Salvador/Warts/Hippo (SWH) pathway and its transcriptional effector Yorkie (Yki). Expression of the Crb intracellular domain elevates Yki activity, and this correlates in tissues and cultured cells with loss of Expanded (Ex), an apically localized SWH component that inhibits Yki. Reciprocally, loss of crb elevates Ex levels, although this excess Ex does not concentrate to its normal location at apical junctions. The Ex-regulatory domain of Crb maps to the juxtamembrane FERM-binding motif (JM), a cytoskeletal interaction domain distinct from the PDZ-binding motif (PBM) through which Crb binds polarity factors. Expression of Crb-JM drives Yki activity and organ growth with little effect on tissue architecture, while Crb-PBM reciprocally produces tissue architectural defects without significant effect on Yki activity.

Conclusions: These studies identify Crb as a novel SWH regulator via JM-dependent effects on Ex levels and localization and suggest that discrete domains within Crb may allow it to integrate junctional polarity signals with a conserved growth pathway.
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http://dx.doi.org/10.1016/j.cub.2010.03.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2855393PMC
April 2010

Functional interactions between the erupted/tsg101 growth suppressor gene and the DaPKC and rbf1 genes in Drosophila imaginal disc tumors.

PLoS One 2009 Sep 29;4(9):e7039. Epub 2009 Sep 29.

Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia, United States of America.

Background: The Drosophila gene erupted (ept) encodes the fly homolog of human Tumor Susceptibility Gene-101 (TSG101), which functions as part of the conserved ESCRT-1 complex to facilitate the movement of cargoes through the endolysosomal pathway. Loss of ept or other genes that encode components of the endocytic machinery (e.g. synatxin7/avalanche, rab5, and vps25) produces disorganized overgrowth of imaginal disc tissue. Excess cell division is postulated to be a primary cause of these 'neoplastic' phenotypes, but the autonomous effect of these mutations on cell cycle control has not been examined.

Principal Findings: Here we show that disc cells lacking ept function display an altered cell cycle profile indicative of deregulated progression through the G1-to-S phase transition and express reduced levels of the tumor suppressor ortholog and G1/S inhibitor Rbf1. Genetic reductions of the Drosophila aPKC kinase (DaPKC), which has been shown to promote tumor growth in other fly tumor models, prevent both the ept neoplastic phenotype and the reduction in Rbf1 levels that otherwise occurs in clones of ept mutant cells; this effect is coincident with changes in localization of Notch and Crumbs, two proteins whose sorting is altered in ept mutant cells. The effect on Rbf1 can also be blocked by removal of the gamma-secretase component presenilin, suggesting that cleavage of a gamma-secretase target influences Rbf1 levels in ept mutant cells. Expression of exogenous rbf1 completely ablates ept mutant eye tissues but only mildly affects the development of discs composed of cells with wild type ept.

Conclusions: Together, these data show that loss of ept alters nuclear cell cycle control in developing imaginal discs and identify the DaPKC, presenilin, and rbf1 genes as modifiers of molecular and cellular phenotypes that result from loss of ept.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0007039PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2739425PMC
September 2009

Genetic interactions between the Drosophila tumor suppressor gene ept and the stat92E transcription factor.

PLoS One 2009 Sep 29;4(9):e7083. Epub 2009 Sep 29.

Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia, United States of America.

Background: Tumor Susceptibility Gene-101 (TSG101) promotes the endocytic degradation of transmembrane proteins and is implicated as a mutational target in cancer, yet the effect of TSG101 loss on cell proliferation in vertebrates is uncertain. By contrast, Drosophila epithelial tissues lacking the TSG101 ortholog erupted (ept) develop as enlarged undifferentiated tumors, indicating that the gene can have anti-growth properties in a simple metazoan. A full understanding of pathways deregulated by loss of Drosophila ept will aid in understanding potential links between mammalian TSG101 and growth control.

Principal Findings: We have taken a genetic approach to the identification of pathways required for excess growth of Drosophila eye-antennal imaginal discs lacking ept. We find that this phenotype is very sensitive to the genetic dose of stat92E, the transcriptional effector of the Jak-Stat signaling pathway, and that this pathway undergoes strong activation in ept mutant cells. Genetic evidence indicates that stat92E contributes to cell cycle deregulation and excess cell size phenotypes that are observed among ept mutant cells. In addition, autonomous Stat92E hyper-activation is associated with altered tissue architecture in ept tumors and an effect on expression of the apical polarity determinant crumbs.

Conclusions: These findings identify ept as a cell-autonomous inhibitor of the Jak-Stat pathway and suggest that excess Jak-Stat signaling makes a significant contribution to proliferative and tissue architectural phenotypes that occur in ept mutant tissues.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0007083PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2747001PMC
September 2009

The archipelago tumor suppressor gene limits rb/e2f-regulated apoptosis in developing Drosophila tissues.

Curr Biol 2009 Sep 3;19(18):1503-10. Epub 2009 Sep 3.

Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA.

Background: The Drosophila archipelago gene (ago) encodes the specificity component of a ubiquitin ligase that targets the cyclin E and dMyc proteins for degradation. Its human ortholog, Fbw7, is commonly lost in cancers, suggesting that failure to degrade ago/Fbw7 targets drives excess tissue growth.

Results: We find that ago loss induces hyperplasia of some organs but paradoxically reduces the size of the adult eye. This reflects a requirement for ago to restrict apoptotic activity of the rbf1/de2f1 pathway adjacent to the eye-specific morphogenetic furrow (MF): ago mutant cells display elevated de2f1 activity, express the prodeath dE2f1 targets hid and rpr, and undergo high rates of apoptosis. These phenotypes are dependent on rbf1, de2f1, hid, and the rbf1/de2f1 regulators cyclin E and dacapo but are independent of dp53. A transactivation-deficient de2f1 allele blocks MF-associated apoptosis of ago mutant cells but does not retard their clonal overgrowth, indicating that intact de2f1 function is required for the death but not overproliferation of ago cells. Epidermal growth factor receptor (EGFR) and wingless (wg) alleles also modify the ago apoptotic phenotype, indicating that these pathways may modulate the underlying sensitivity of ago mutant cells to apoptotic signals.

Conclusions: These data show that ago loss requires a collaborating block in cell death to efficiently drive tissue overgrowth and that this conditional phenotype reflects a role for ago in restricting apoptotic output of the rbf1/de2f1 pathway. Moreover, the susceptibility of ago mutant cells to succumb to this apoptotic program appears to depend on local variations in extracellular signaling that could thus determine tissue-specific fates of ago mutant cells.
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http://dx.doi.org/10.1016/j.cub.2009.07.068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2755617PMC
September 2009
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