Elizabeth Silva

The Tumor-Suppressor Gene fat Controls Tissue Growth Upstream of Expanded in the Hippo Signaling Pathway

BACKGROUND The tight control of cell proliferation and cell death is essential to normal tissue development, and the loss of this control is a hallmark of cancers. Cell growth and cell death are coordinately regulated during development by the Hippo signaling pathway. The Hippo pathway consists of the Ste20 family kinase Hippo, the WW adaptor protein Salvador, and the NDR kinase Warts. Loss of Hippo signaling in Drosophila leads to enhanced cell proliferation and decreased apoptosis, resulting in massive tissue overgrowth through increased expression of targets such as Cyclin E and Diap1. The cytoskeletal proteins Merlin and Expanded colocalize at apical junctions and function redundantly upstream of Hippo. It is not clear how they regulate growth or how they are localized to apical junctions. RESULTS We find that another Drosophila tumor-suppressor gene, the atypical cadherin fat, regulates both cell proliferation and cell death in developing imaginal discs. Loss of fat leads to increased Cyclin E and Diap1 expression, phenocopying loss of Hippo signaling. Ft can regulate Hippo phosphorylation, a measure of its activation, in tissue culture. Importantly, fat is needed for normal localization of Expanded at apical junctions in vivo. Genetic-epistasis experiments place fat with expanded in the Hippo pathway. CONCLUSIONS Together, these data suggest that Fat functions as a cell-surface receptor for the Expanded branch of the conserved Hippo growth control pathway.

Undertaker, a Drosophila Junctophilin, links Draper-mediated phagocytosis and calcium homeostasis.

Phagocytosis is important during development and in the immune response for the removal of apoptotic cells and pathogens, yet its molecular mechanisms are poorly understood. In Caenorhabditis elegans, the CED2/5/10/12 pathway regulates actin during phagocytosis of apoptotic cells, whereas the role of the CED1/6/7 pathway in phagocytosis is unclear. We report that Undertaker (UTA), a Drosophila Junctophilin protein, is required for Draper (CED-1 homolog)-mediated phagocytosis. Junctophilins couple Ca2+ channels at the plasma membrane to those of the endoplasmic reticulum (ER), the Ryanodine receptors. We place Draper, its adaptor drCed-6, UTA, the Ryanodine receptor Rya-r44F, the ER Ca2+ sensor dSTIM, and the Ca2+-release-activated Ca2+ channel dOrai in the same pathway that promotes calcium homeostasis and phagocytosis. Thus, our results implicate a Junctophilin in phagocytosis and link Draper-mediated phagocytosis to Ca2+ homeostasis, highlighting a previously uncharacterized role for the CED1/6/7 pathway.

ATM is required for telomere maintenance and chromosome stability during Drosophila development.

ATM is a large, multifunctional protein kinase that regulates responses required for surviving DNA damage: including DNA repair, apoptosis, and cell cycle checkpoints. Here, we show that Drosophila ATM function is essential for normal adult development. Extensive, inappropriate apoptosis occurs in proliferating atm mutant tissues, and in clonally derived atm mutant embryos, frequent mitotic defects were seen. At a cellular level, spontaneous telomere fusions and other chromosomal abnormalities are common in atm larval neuroblasts, suggesting a conserved and essential role for dATM in the maintenance of normal telomeres and chromosome stability. Evidence from other systems supports the idea that DNA double-strand break (DSB) repair functions of ATM kinases promote telomere maintenance by inhibition of illegitimate recombination or fusion events between the legitimate ends of chromosomes and spontaneous DSBs. Drosophila will be an excellent model system for investigating how these ATM-dependent chromosome structural maintenance functions are deployed during development. Because neurons appear to be particularly sensitive to loss of ATM in both flies and humans, this system should be particularly useful for identifying cell-specific factors that influence sensitivity to loss of dATM and are relevant for understanding the human disease, ataxia-telangiectasia.

Phosphorylation of the Tumor Suppressor Fat Is Regulated by Its Ligand Dachsous and the Kinase Discs Overgrown

The Drosophila tumor suppressor gene fat encodes a large cadherin that regulates growth and a form of tissue organization known as planar cell polarity (PCP). Fat regulates growth via the Hippo kinase pathway, which controls expression of genes promoting cell proliferation and inhibiting apoptosis (reviewed in). The Hippo pathway is highly conserved and is implicated in the regulation of mammalian growth and cancer development. Genetic studies suggest that Fat activity is regulated by binding to another large cadherin, Dachsous (Ds). The tumor suppressor discs overgrown (dco)/Casein Kinase I delta/epsilon also regulates Hippo activity and PCP. The biochemical nature of how Fat, Ds, and Dco interact to regulate these pathways is poorly understood. Here we demonstrate that Fat is cleaved to generate 450 kDa and 110 kDa fragments (Fat(450) and Fat(110)). Fat(110) contains the cytoplasmic and transmembrane domain. The cytoplasmic domain of Fat binds Dco and is phosphorylated by Dco at multiple sites. Importantly, we show Fat forms cis-dimers and that Fat phosphorylation is regulated by Dachsous and Dco in vivo. We propose that Ds regulates Dco-dependent phosphorylation of Fat and Fat-associated proteins to control Fat signaling in growth and PCP.

Extracellular glutamate, aspartate and arginine increase in the ventral posterolateral thalamic nucleus during nociceptive stimulation.

Although there is evidence that the thalamus plays a remarkable role in pain processing few in vivo studies on the thalamic neurochemical correlates of pain have been done. In the present experiments a combination of capillary zone electrophoresis with laser-induced fluorescence detection (CZE-LIF) and microdialysis in freely moving rats was used to measure extracellular arginine, glutamate and aspartate in the thalamus during the formalin test. Microdialysis probes were implanted in the left ventral posterolateral (VPL) nucleus of the thalamus in rats. Samples were collected every 30 s, derivatized with fluorescein isothyocyanate and injected into a CZE-LIF instrument. After nine baseline samples, a subcutaneous formalin (5%, 50 microl) injection in the right hind paw caused an increase of arginine, glutamate and aspartate that lasted for about 3 min. These increases were calcium and nerve impulse dependent. These results indicate that the release of arginine, glutamate and aspartate may mediate rapid pain neural transmission in the VPL nucleus of the thalamus.

Noxious stimulation increases glutamate and arginine in the periaqueductal gray matter in rats: a microdialysis study

&NA; The periaqueductal gray matter (PAG) is an important center in the modulation of behavioral responses during nociception and stress. In the present experiment, extracellular excitatory amino acid overflow in the PAG was measured every 30 s during noxious stimulation. A combination of in vivo brain microdialysis in freely moving rats and capillary zone electrophoresis with laser induced‐fluorescence detection allowed us to detect short lasting changes of excitatory amino acid in dialysates. A formalin injection in the hindpaw of the rat increased glutamate, arginine and aspartate concentration in PAG dialysates. This increase was calcium and nerve impulse‐dependent, suggesting neuronal and glial origin of glutamate and arginine, respectively. Handling, pinching or saline injection in the hind paw did not increase glutamate showing that this neurochemical phenomenon is related to painful and persistent noxious stimulation. The results suggest that a rapid excitation of the PAG occurs during noxious stimulation. The role of glutamate and arginine in analgesia is discussed.

Essential genes in proximal 3L heterochromatin of Drosophila melanogaster.

Abstract. We have further characterized essential loci within the centric heterochromatin of the left arm of chromosome 3 (3L) of Drosophila melanogaster, using EMS, radiation and P element mutagenesis. We failed to find any new essential genes, a result that suggests a lower-than-average gene density in this region. Mutations affecting expression of the most proximal gene [lethal 1, l1 or l(3)80Fj] act as dominant suppressors of Polycomb (Pc), behavior which is consistent with a putative trithorax group (trx-G) gene. The third gene to the left of the centromere [lethal 3, l3 or l(3)80Fh] is likely to correspond to verthandi (vtd), a known trx-G gene that plays a role in the regulation of hedgehog (hh) expression and signalling. The intervening gene [lethal 2, l2 or l(3)80Fi] is required throughout development, and mutant alleles have interesting phenotypes; in various allelic combinations that survive, we observe fertility, bristle, wing, eye and cuticle defects.

Nitric oxide synthase is not essential for Drosophila development

Nitric oxide (NO) is a key regulator of diverse biological processes, including the modulation of blood vessel tone [1]. Nitric oxide synthase (NOS), which oxidizes arginine to produce NO and citrulline [2], is found in organisms from bacteria to humans. Despite the impact of NO on physiology, mice lacking all three mammalian NOS isoforms develop to term and are viable [3]. There is a single NOS ortholog encoded in the Drosophila genome (Nos). Regulski et al. [4] described a mutation in a conserved residue that abrogates NOS activity, and reported that this lesion confers lethality (NosC). However, two lines of evidence led us to believe that this lethality could be due to a closely associated mutation rather than the lesion in Nos itself. First, the lethality was not rescued by reintroduction of NOS. Second, while the authors convincingly demonstrate that they have generated a mutation in the Nos gene that inactivates the enzyme, they do so for only one of the 17 alleles that they assign to the Nos complementation group. Beginning with a stock of NosC provided by Regulski et al. [4], we isolated recombinant chromosomes in which we separated the lethal lesion from the point mutation in NosC. Additionally, we generated a deletion that removes significant portions of the Nos coding sequences, including those responsible for synthesis of NO, and found it to be homozygous viable. Both our deletion and NosC eliminate NOS enzymatic activity without affecting Drosophila development, and without obviously compromising the health of the flies.

Essential genes in autosomal heterochromatin of Drosophila melanogaster.

We are taking two approaches to understanding the structure, function and regulation of essential genes within Drosophilaheterochromatin. In the first, we have undertaken a genetic and molecular characterization of essential genes within proximal 3L heterochromatin. The expression of such ‘resident’ genes within a heterochromatic environment is paradoxical and poorly understood, given that the same environment can inactivate euchromatic sequences (position effect variegation, or PEV). A second approach involves the study of the local chromosomal environment of heterochromatic (het) genes, as assayed both biochemically, and via the effects of genetic modifiers of PEV, the latter being putative components important for het gene expression. Our results to date suggest that the three most proximal genes in 3L heterochromatin have key roles in development, and indicate strong effects of combinations of genetic modifiers of PEV on het gene expression.

Ensuring transparency and minimization of methodologic bias in preclinical pain research: PPRECISE considerations

Abstract There is growing concern about lack of scientific rigor and transparent reporting across many preclinical fields of biological research. Poor experimental design and lack of transparent reporting can result in conscious or unconscious experimental bias, producing results that are not replicable. The Analgesic, Anesthetic, and Addiction Clinical Trial Translations, Innovations, Opportunities, and Networks (ACTTION) public–private partnership with the U.S. Food and Drug Administration sponsored a consensus meeting of the Preclinical Pain Research Consortium for Investigating Safety and Efficacy (PPRECISE) Working Group. International participants from universities, funding agencies, government agencies, industry, and a patient advocacy organization attended. Reduction of publication bias, increasing the ability of others to faithfully repeat experimental methods, and increased transparency of data reporting were specifically discussed. Parameters deemed essential to increase confidence in the published literature were clear, specific reporting of an a priori hypothesis and definition of primary outcome measure. Power calculations and whether measurement of minimal meaningful effect size to determine these should be a core component of the preclinical research effort provoked considerable discussion, with many but not all agreeing. Greater transparency of reporting should be driven by scientists, journal editors, reviewers, and grant funders. The conduct of high-quality science that is fully reported should not preclude novelty and innovation in preclinical pain research, and indeed, any efforts that curtail such innovation would be misguided. We believe that to achieve the goal of finding effective new treatments for patients with pain, the pain field needs to deal with these challenging issues.