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2003

A new genetic locus controlling growth and proliferation in Drosophila melanogaster.

Author(s) : Raisin S, Pantalacci S, Breittmayer J, Leopold P,
Journal : Genetics
2003
Multicellular organisms grow through both proliferation and growth of their individual cells. We have conducted a P-element-based misexpression screen for genes whose upregulation alters wing disc growth during development. One particular group of four P elements, all inserted at cytological location 61C7-8, exhibited specific overgrowth upon misexpression in proliferating imaginal tissues. Clonal analysis revealed that upon misexpression, cell number was increased but cell size was not affected, indicating that cell growth and proliferation were induced in a coordinate manner. Loss of function at the locusproduced small flies with reduced cell number, consistent with the presence of agene encoding a positive growth regulator. We characterized a new transcription unit initiating in a region adjacent to the P insertions, which generated a complex series of polyadenylated transcripts. Although these RNAs were induced in response to misexpression, none was sufficient by itself to recapitulate overgrowth when overexpressed. This suggested either that a particular combination of these transcripts was necessary or that other sequences are involved.

A nutrient sensor mechanism controls Drosophila growth.

Author(s) : Colombani J, Raisin S, Pantalacci S, Radimerski T, Montagne J, Leopold P,
Journal : Cell
2003
Organisms modulate their growth according to nutrient availability. Although individual cells in a multicellular animal may respond directly to nutrient levels, growth of the entire organism needs to be coordinated. Here, we provide evidence that in Drosophila, coordination of organismal growth originates from the fat body, an insect organ that retains endocrine and storage functions of the vertebrate liver. In a genetic screen for growth modifiers, we identified slimfast, a gene that encodes an amino acid transporter. Remarkably, downregulation of slimfast specifically within the fat body causes a global growth defect similar to that seen in Drosophila raised under poor nutritional conditions. This involves TSC/TOR signaling in the fat body, and a remote inhibition of organismal growth via local repression of PI3-kinase signaling in peripheral tissues. Our results demonstrate that the fat body functions as a nutrient sensor that restricts global growth through a humoral mechanism.

A sensitized genetic screen to identify novel regulators and components of the Drosophila janus kinase/signal transducer and activator of transcription pathway.

Author(s) : Bach E, Vincent S, Zeidler M, Perrimon N,
Journal : Genetics
2003
The JAK/STAT pathway exerts pleiotropic effects on a wide range of developmentalprocesses in Drosophila. Four key components have been identified: Unpaired, a secreted ligand; Domeless, a cytokine-like receptor; Hopscotch, a JAK kinase; and Stat92E, a STAT transcription factor. The identification of additional components and regulators of this pathway remains an important issue. To this end, we have generated a transgenic line where we misexpress the upd ligand in the developingDrosophila eye. GMR-upd transgenic animals have dramatically enlarged eye-imaginal discs and compound eyes that are normally patterned. We demonstratethat the enlarged-eye phenotype is a result of an increase in cell number, and not cell volume, and arises from additional mitoses in larval eye discs. Thus, the GMR-upd line represents a system in which the proliferation and differentiation of eye precursor cells are separable. Removal of one copy of stat92E substantially reduces the enlarged-eye phenotype. We performed an F1 deficiency screen to identify dominant modifiers of the GMR-upd phenotype. We have identified 9 regions that enhance this eye phenotype and two specific enhancers: C-terminal binding protein and Daughters against dpp. We also identified 20 regions that suppress GMR-upd and 13 specific suppressors: zeste-white 13, pineapple eye, Dichaete, histone 2A variant, headcase, plexus, kohtalo, crumbs, hedgehog, decapentaplegic, thickveins, saxophone, and Mothers against dpp.

Arsenic enhances the activation of Stat1 by interferon gamma leading to synergistic expression of IRF-1.

Author(s) : Chelbi-alix M, Bobe P, Benoit G, Canova A, Pine R,
Journal : Oncogene
2003
Arsenic trioxide (As2O3) can induce clinical remission in patients with acute promyelocytic leukemia (APL), including those who have relapsed after treatment with all-trans-retinoic acid (RA). In vitro studies with the APL-derived NB4 cell line showed that As2O3 exerts a dose-dependent dual effect, which induces apoptosis at 1 microM, whereas at a lower concentration of 0.1 microM, a partialdifferentiation of APL is observed. In non-APL cells, interferon (IFN) alpha and1 microM As2O3 act synergistically to induce apoptosis. In this report, we show that in NB4 cells and in two RA-resistant NB4-derived cell lines, NB4-R1 and NB4-R2, IFNalpha or IFNgamma combined with 0.1 microM As2O3 lead to an increasedmaturation effect. Moreover, IFNgamma alone is able to differentiate RA-sensitive and -resistant cells with a higher maturation effect on NB4-R2 cells. In contrast, all these cells underwent apoptosis in the presence of the cytokine and a higher concentration of As2O3. IFNgamma boosted As2O3-induced apoptosis in APLcells as tested by TUNEL, Annexin V staining and activation of caspase 3. As2O3 differently altered IFN-induced gene products; it downregulated PML/RARalpha andPML, did not alter PKR and Stat1, and upregulated interferon regulatory family (IRF)-1. Synergism by IFNgamma and arsenic on IRF-1 expression is mediated by a composite element in the IRF-1 promoter that includes an IFNgamma-activation site (GAS) overlapped by a nonconsensus site for nuclear factor kappa B (NFkappaB). Arsenic has no effect on NFkappaB, whereas it enhances the activation of Stat1 by IFNgamma in NB4 cells leading to an increase in IRF-1 expression.

Hsk1-Dfp1 is required for heterochromatin-mediated cohesion at centromeres.

Author(s) : Bailis J, Bernard P, Antonelli R, Allshire R, Forsburg S,
Journal : Nat Cell Biol
2003
Heterochromatin performs a central role in chromosome segregation and stability by promoting cohesion at centromeres. Establishment of both heterochromatin-mediated silencing and cohesion requires passage through S phase, although the mechanism is unknown. Here we demonstrate that Schizosaccharomyces pombe Hsk1 (CDC7), a conserved Dbf4-dependent protein kinase (DDK) that regulates replication initiation, interacts with and phosphorylates the heterochromatin protein 1 (HP1) equivalent Swi6 (ref. 6). Hsk1 and its regulatory subunit Dfp1 function downstream of Swi6 localization to promote heterochromatin function andcohesion specifically at centromeres. This role for Hsk1-Dfp1 is separable from its replication initiation activity, providing a temporal link between S phase and centromere cohesion that is mediated by heterochromatin.

Nurr1 regulates dopamine synthesis and storage in MN9D dopamine cells.

Author(s) : Hermanson E, Joseph B, Castro D, Lindqvist E, Aarnisalo P, Wallen A, Benoit G, Hengerer B, Olson L, Perlmann T,
Journal : Exp Cell Res
2003
Nurr1, a transcription factor belonging to the nuclear receptor family, is essential for the generation of midbrain dopamine (DA) cells during embryonic development. Nurr1 continues to be expressed in adult DA neurons but the role for Nurr1 in inducing and regulating basic dopaminergic functions such as dopamine synthesis and storage has remained unknown. We have previously used MN9D dopamine cells to analyze the role of Nurr1 and retinoids in DA cell maturation. These studies demonstrated that both Nurr1 and retinoids induce cell cycle arrest and a mature morphology. Here we used MN9D cells to investigate how Nurr1 regulates dopaminergic functions. Our results demonstrate that Nurr1, but not retinoids, increases DA content and the expression of aromatic L-amino acid decarboxylase (AADC) and vesicular monoamine transporter-2 (VMAT2) in MN9D cells. In a Nurr1-inducible cell line upregulation of VMAT2 is dependent on continuous Nurr1expression. Moreover, AADC and VMAT2 are deregulated in midbrain DA cells of Nurr1 knockout embryos as revealed by in situ hybridization. Together, the results provide evidence indicating an instructive role for Nurr1 in controllingDA synthesis and storage.

p57(Kip2) cooperates with Nurr1 in developing dopamine cells.

Author(s) : Joseph B, Wallen-Mackenzie A, Benoit G, Murata T, Joodmardi E, Okret S, Perlmann T,
Journal : Proc Natl Acad Sci U S A
2003
Cyclin-dependent kinase inhibitors of the Cip/Kip family play critical roles in regulating cell proliferation during embryogenesis. However, these proteins alsoinfluence cell differentiation by mechanisms that have remained unknown. Here weshow that p57Kip2 is expressed in postmitotic differentiating midbrain dopamine cells. Induction of p57Kip2 expression depends on Nurr1, an orphan nuclear receptor that is essential for dopamine neuron development. Moreover, analyses of p57Kip2 gene-targeted mice revealed that p57Kip2 is required for the maturation of midbrain dopamine neuronal cells. Additional experiments in a dopaminergic cell line demonstrated that p57Kip2 can promote maturation by a mechanism that does not require p57Kip2-mediated inhibition of cyclin-dependent kinases. Instead, evidence indicates that p57Kip2 functions by a direct protein-protein interaction with Nurr1. Thus, in addition to its established function in controlof proliferation, these results reveal a mechanism whereby p57Kip2 influences postmitotic differentiation of dopamine neurons.

Structure and function of Nurr1 identifies a class of ligand-independent nuclear receptors.

Author(s) : Wang Z, Benoit G, Liu J, Prasad S, Aarnisalo P, Liu X, Xu H, Walker N, Perlmann T,
Journal : Nature
2003
Members of the nuclear receptor (NR) superfamily of transcription factors modulate gene transcription in response to small lipophilic molecules. Transcriptional activity is regulated by ligands binding to the carboxy-terminalligand-binding domains (LBDs) of cognate NRs. A subgroup of NRs referred to as 'orphan receptors' lack identified ligands, however, raising issues about the function of their LBDs. Here we report the crystal structure of the LBD of the orphan receptor Nurr1 at 2.2 A resolution. The Nurr1 LBD adopts a canonical protein fold resembling that of agonist-bound, transcriptionally active LBDs in NRs, but the structure has two distinctive features. First, the Nurr1 LBD contains no cavity as a result of the tight packing of side chains from several bulky hydrophobic residues in the region normally occupied by ligands. Second, Nurr1 lacks a 'classical' binding site for coactivators. Despite these differences, the Nurr1 LBD can be regulated in mammalian cells. Notably, transcriptional activity is correlated with the Nurr1 LBD adopting a more stableconformation. Our findings highlight a unique structural class of NRs and definea model for ligand-independent NR function.

The MEK-1/ERKs signalling pathway is differentially involved in the self-renewal of early and late avian erythroid progenitor cells.

Author(s) : Dazy S, Damiola F, Parisey N, Beug H, Gandrillon O,
Journal : Oncogene
2003
Making decisions between self-renewal and differentiation is a central ability of stem cells. Elucidation of molecular networks governing this decision is therefore of prime importance. A model of choice to explore this question is represented by chicken erythroid progenitors, in which self-renewal versus differentiation as well as progenitor maturation are regulated by external factor combinations. We used this system to study whether similar or different signalling pathways were involved in the self-renewal of early, immature or moremature erythroid progenitors. We show that a transforming growth factor (TGF)-alpha-activated Ras/MEK-1/ERK1/2 pathway is strictly required for immatureself-renewing cells but becomes fully dispensable when those cells are induced to differentiate. Consequently, pharmacological inhibition of this pathway led to spontaneous differentiation, only dependent on the presence of survival signals.Conversely, ectopic expression of a constitutive form of MEK-1 stimulates renewal and arrests differentiation process. Finally, we demonstrate that the ERK/MAPK signalling pathway is required in early but not in late primary erythroid progenitors, which can be turned into each other by different growth factor combinations specifically driving their renewal. To the best of our knowledge, this is the first description of a central role of ERK/MAPK signalling in regulating progenitor plasticity in the same cell type under different environmental conditions.

The Salvador partner Hippo promotes apoptosis and cell-cycle exit in Drosophila.

Author(s) : Pantalacci S, Tapon N, Leopold P,
Journal : Nat Cell Biol
2003
Tissue growth during animal development is tightly controlled so that the organism can develop harmoniously. The salvador (sav) gene, which encodes a scaffold protein, has been shown to restrict cell number by coordinating cell-cycle exit and apoptosis during Drosophila development. Here we identify Hippo (Hpo), the Drosophila orthologue of the mammalian MST1 and MST2 serine/threonine kinases, as a partner of Sav. Loss of hpo function leads to sav-like phenotypes, whereas gain of hpo function results in the opposite phenotype. Whereas Sav and Hpo normally restrict cellular quantities of the Drosophila inhibitor of apoptosis protein DIAP1, overexpression of Hpo destabilizes DIAP1 in cell culture. We show that DIAP1 is phosphorylated in a Hpo-dependent manner in S2 cells and that Hpo can phosphorylate DIAP1 in vitro. Thus, Hpo may promote apoptosis by reducing cellular amounts of DIAP1. In addition, we show that Sav is an unstable protein that is stabilized by Hpo. We propose that Hpo and Sav function together to restrict tissue growth in vivo.