Tay Bridge Is a Negative Regulator of EGFR Signalling and Interacts with Erk and Mkp3 in theDrosophila melanogaster Wing

The regulation of Extracellular regulated kinase (Erk) activity is a key aspect of signalling by pathways activated by extracellular ligands acting through tyrosine kinase transmembrane receptors. In this process, participate proteins with kinase activity that phosphorylate and activate Erk, as well as different phosphatases that inactivate Erk by de-phosphorylation. The state of Erk phosphorylation affects not only its activity, but also its subcellular localization, defining the repertoire of Erk target proteins, and consequently, the cellular response to Erk. In this work, we characterise Tay bridge as a novel component of the EGFR/Erk signalling pathway. Tay bridge is a large nuclear protein with a domain of homology with human AUTS2, and was previously identified due to the neuronal phenotypes displayed by loss-of-function mutations. We show that Tay bridge antagonizes EGFR signalling in the Drosophila melanogaster wing disc and other tissues, and that the protein interacts with both Erk and Mkp3. We suggest that Tay bridge constitutes a novel element involved in the regulation of Erk activity, acting as a nuclear docking for Erk that retains this protein in an inactive form in the nucleus. © 2013 Molnar, de Celis.BFU2009-09403; BFU2012-33994; CSD2007-00008; Fundación Ramón ArecesPeer Reviewe

The balance between GMD and OFUT1 regulates notch signaling pathway activity by modulating notch stability

The Notch signaling pathway plays an important role in development and physiology. In Drosophila, Notch is activated by its Delta or Serrate ligands, depending in part on the sugar modifications present in its extracellular domain. O-fucosyltransferase-1 (OFUT1) performs the first glycosylation step in this process, O-fucosylating various EGF repeats at the Notch extracellular domain. Besides its O-fucosyltransferase activity, OFUT1 also behaves as a chaperone during Notch synthesis and is able to down regulate Notch by enhancing its endocytosis and degradation. We have reevaluated the roles that O-fucosylation and the synthesis of GDP-fucose play in the regulation of Notch protein stability. Using mutants and the UAS/Gal4 system, we modified in developing tissues the amount of GDP-mannosedeshydratase (GMD), the first enzyme in the synthesis of GDP-fucose. Our results show that GMD activity, and likely the levels of GDPfucose and O-fucosylation, are essential to stabilize the Notch protein. Notch degradation observed under low GMD expression is absolutely dependent on OFUT1 and this is also observed in Notch Abruptex mutants, which have mutations in some potential O-fucosylated EGF domains. We propose that the GDP-fucose/OFUT1 balance determines the ability of OFUT1 to endocytose and degrade Notch in a manner that is independent of the residues affected by Abruptex mutations in Notch EGF domains.This work was funded by ICM P06-039F grant to A.G. and by a BFU2009-09403 grant of the M.E.C. to J.F.dC. An institutional grant from the Ramón Areces Foundation to the CBMSO is also acknowledged.Peer Reviewe

A refutation to 'A new A-P compartment boundary and organizer in holometabolous insect wings'.

We respond to a recent report by Abbasi and Marcus who present two main findings: first they argue that there is an organiser and a compartment boundary within the posterior compartment of the butterfly wing. Second, they present evidence for a previously undiscovered lineage boundary near wing vein 5 in Drosophila, a boundary that delineates a "far posterior" compartment. Clones of cells were marked with the yellow mutation and they reported that these clones always fail to cross a line close to vein 5 on the Drosophila wing. In our hands yellow proved an unusable marker for clones in the wing blade and therefore we reexamined the matter. We marked clones of cells with multiple wing hairs or forked and found a substantial proportion of these clones cross the proposed lineage boundary near vein 5, in conflict with their findings and conclusion. As internal controls we showed that these same clones respect the other two well established compartment boundaries: the anteroposterior compartment boundary is always respected. The dorsoventral boundary is mostly respected, and is crossed only by clones that are induced early in development, consistent with many reports. We question the validity of Abbasi and Marcus' conclusions regarding the butterfly wing but present no new data.Arising from: R. Abbasi and J. M. Marcus Sci. Rep. 7, 16337 (2017); https://doi.org/10.1038/s41598-017-16553-5 .WELLCOME TRUST 107060/Z/15/

Drosophila as a model system for genetic and genomic research

The sequencing of the Drosophila genome allowed the identification of most coding sequences, highlighting the necessity for a functional assignation of the identified genes. The information extracted from the sequence directly classified a considerable fraction of genes into known molecular categories, although there is still a large proportion of them that, due to poor sequence conservation, are not included into any informative class. Furthermore, in many instances the molecular nature of a protein is not particularly revealing about its functional requirements and network of interactions. In this manner, complementary genomic approaches to gene identification by sequence conservation are fundamental both in Drosophila and other organisms to assign particular functions to annotated genes. The approach more successful in the Drosophila field is the undertaking of genetic screenings to identify sets of interacting genes and genes controlling particular cellular processes. Classic genetic screens comprise all those based on a “phenotypic” paradigm, where the generation of large collections of mutant chromosomes is followed by their mapping. This approach has been recently expanded to include “genomic” tools, such as the use of microarrays and interference RNA, as well as reverse-genetics techniques, seeding the way to a “functional” annotation of the Drosophila genome.Peer reviewe

MEIS1, PREP1, and PBX4 Are Differentially Expressed in Acute Lymphoblastic Leukemia: Association of MEIS1 Expression with Higher Proliferation and Chemotherapy Resistance

<p>Abstract</p> <p>Background</p> <p>The Three-amino acid-loop-extension (<it>TALE</it>) superfamily of homeodomain-containing transcription factors have been implicated in normal hematopoiesis and in leukemogenesis and are important survival, differentiation, and apoptosis pathway modulators. In this work, we determined the expression levels of <it>TALE </it>genes in leukemic-derived cell lines, in blood samples of patients with Acute lymphoblastic leukemia (ALL), and in the blood samples of healthy donors.</p> <p>Results</p> <p>Here we show increased expression of <it>MEIS1, MEIS2, </it>and <it>PREP1 </it>genes in leukemia-derived cell lines compared with blood normal cells. High levels of <it>MEIS1 </it>and <it>PREP1</it>, and low levels of <it>PBX4 </it>expression were also founded in samples of patients with ALL. Importantly, silencing of <it>MEIS1 </it>decreases the proliferation of leukemia-derived cells but increases their survival after etoposide treatment. Etoposide-induced apoptosis induces down-regulation of MEIS1 expression or <it>PREP1 </it>up-regulation in chemotherapy-resistant cells.</p> <p>Conclusions</p> <p>Our results indicate that up-regulation of <it>MEIS1 </it>is important for sustaining proliferation of leukemic cells and that down-regulation of <it>MEIS1 </it>or up-regulation of <it>PREP1 </it>and <it>PBX </it>genes could be implicated in the modulation of the cellular response to chemotherapeutic-induced apoptosis.</p

Independent roles of Drosophila Moesin in imaginal disc morphogenesis and hedgehog signalling

The three ERM proteins (Ezrin, Radixin and Moesin) form a conserved family required in many developmental processes involving regulation of the cytoskeleton. In general, the molecular function of ERM proteins is to link specific membrane proteins to the actin cytoskeleton. In Drosophila, loss of moesin (moe) activity causes incorrect localisation of maternal determinants during oogenesis, failures in rhabdomere differentiation in the eye and alterations of epithelial integrity in the wing imaginal disc. Some aspects of Drosophila Moe are related to the activity of the small GTPase RhoA, because the reduction of RhoA activity corrects many phenotypes of moe mutant embryos and imaginal discs. We have analysed the phenotype of moesin loss-of-function alleles in the wing disc and adult wing, and studied the effects of reduced Moesin activity on signalling mediated by the Notch, Decapentaplegic, Wingless and Hedgehog pathways. We found that reductions in Moesin levels in the wing disc cause the formation of wing-tissue vesicles and large thickenings of the vein L3, corresponding to breakdowns of epithelial continuity in the wing base and modifications of Hedgehog signalling in the wing blade, respectively. We did not observe any effect on signalling pathways other than Hedgehog, indicating that the moe defects in epithelial integrity have not generalised effects on cell signalling. The effects of moe mutants on Hedgehog signalling depend on the correct gene-dose of rhoA, suggesting that the requirements for Moesin in disc morphogenesis and Hh signalling in the wing disc are mediated by its regulation of RhoA activity. The mechanism linking Moesin activity with RhoA function and Hedgehog signalling remains to be elucidatedGrants from Dirección General de Investigación Científica y Técnica (BCM2003-1191 and GEN2001-4846-C05-01) to J.F.d C. and an institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular “Severo Ochoa” are also acknowledged.Peer reviewe

Regulation of decapentaplegic expression during Drosophila wing veins pupal development

The differentiation of veins in the Drosophila wing relies on localised expression of decapentaplegic (dpp) in pro-vein territories during pupal development. The expression of dpp in the pupal veins requires the integrity of the shortvein region (shv), localised 5′ to the coding region. It is likely that this DNA integrates positive and negative regulatory signals directing dpp transcription during pupal development. Here, we identify a minimal 0.9 kb fragment giving localised expression in the vein L5 and a 0.5 kb fragment giving expression in all longitudinal veins. Using a combination of in vivo expression of reporter genes regulated by shv sequences, in vitro binding assays and sequence comparisons between the shv region of different Drosophila species, we found binding sites for the vein-specific transciption factors Araucan, Knirps and Ventral veinless, as well as binding sites for the Dpp pathway effectors Mad and Med. We conclude that conserved vein-specific enhancers regulated by transcription factors expressed in individual veins collaborate with general vein and intervein regulators to establish and maintain the expression of dpp confined to the veins during pupal developmentA postdoctoral short-term fellowship from the European Molecular Biology Organization (EMBO) to S. S. is acknowledged. Grants from Dirección General de Investigación Científica y Técnica to James Castelli-Gair (BFU2004-01069/BMC), to J.F.d C. (BCM2003-1191 and GEN2001-4846-C05-01), as well as an institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular “Severo Ochoa” are also acknowledgedPeer reviewe

Regulation and function of Spalt proteins during animal development

The genes of the spalt (sal) family play fundamental roles during animal development. The two members of this family in Drosophila, spalt (sal) and spalt-related (salr) encode Zn-finger transcription factors that link the Decapentaplegic (Dpp)/BMP signalling pathway to the patterning of the wing. They are regulated by the Dpp pathway in the wing disc, and they were shown to mediate some of the morphogenetic activities of the Dpp/BMP4 secreted ligand. The sal genes were initially found by virtue of mutations that produce homeotic transformations in the head and tail of the Drosophila embryo. Since then, a number of other requirements have been associated to these genes in Drosophila, including morphogenesis of the respiratory system, cell fate specification of sensory organs and the differentiation of several photoreceptor cells, among others. Vertebrate sal orthologues (spalt-like/sall) have also important developmental roles during neural development and organogenesis, and at least two human sall genes are linked to the genetic diseases Townes Brocks Syndrome (TBS; SALL1 ) and Okihiro Syndrome (OS; SALL4 ). In this review, we will summarize the main characteristics of the sall genes and proteins, pointing out to the similarities in their developmental roles during Drosophila and vertebrate development.Sánchez. J.F.dC. is supported by an institutional grant from the Ramón Areces Foundation to the Centro de Biología Molecular “Severo Ochoa” and by the Ministerio de Educación y Ciencia (BFU2006-06501). R.B. belongs to the Cajal Program (MEC) and is recipient of grants from the Spanish Ministry of Education (BFU2005-00257), the Department of Industry, Tourism and Trade of the Government of the Autonomous Community of the Basque Country (Etortek Research Programs 2005/2006) and from the Innovation Technology Department of the Bizkaia County.Peer reviewe

A refutation to ‘A new A-P compartment boundary and organizer in holometabolous insect wings’

We respond to a recent report by Abbasi and Marcus who present two main findings: first they argue that there is an organiser and a compartment boundary within the posterior compartment of the butterfly wing. Second, they present evidence for a previously undiscovered lineage boundary near wing vein 5 in Drosophila, a boundary that delineates a “far posterior” compartment. Clones of cells were marked with the yellow mutation and they reported that these clones always fail to cross a line close to vein 5 on the Drosophila wing. In our hands yellow proved an unusable marker for clones in the wing blade and therefore we reexamined the matter. We marked clones of cells with multiple wing hairs or forked and found a substantial proportion of these clones cross the proposed lineage boundary near vein 5, in conflict with their findings and conclusion. As internal controls we showed that these same clones respect the other two well established compartment boundaries: the anteroposterior compartment boundary is always respected. The dorsoventral boundary is mostly respected, and is crossed only by clones that are induced early in development, consistent with many reports. We question the validity of Abbasi and Marcus’ conclusions regarding the butterfly wing but present no new data. Arising from: R. Abbasi and J. M. Marcus Sci. Rep. 7, 16337 (2017); https://doi.org/10.1038/s41598-017-16553-5.Ministerio de Economia y Competitivida

Relationships between extramacrochaetae and Notch signalling in Drosophila wing development

The function of extramacrochaetae is required during the development of the Drosophila wing in processes such as cell proliferation and vein differentiation. extramacrochaetae encodes a transcription factor of the HLH family, but unlike other members of this family, Extramacrochaetae lacks the basic region that is involved in interaction with DNA. Some phenotypes caused by extramacrochaetae in the wing are similar to those observed when Notch signalling is compromised. Furthermore, maximal levels of extramacrochaetae expression in the wing disc are restricted to places where Notch activity is higher, suggesting that extramacrochaetae could mediate some aspects of Notch signalling during wing development. We have studied the relationships between extramacrochaetae and Notch in wing development, with emphasis on the processes of vein formation and cell proliferation. We observe strong genetic interaction between extramacrochaetae and different components of the Notch signalling pathway, suggesting a functional relationship between them. We show that the higher level of extramacrochaetae expression coincides with the domain of expression of Notch and its downstream gene Enhancer of split-m(beta). The expression of extramacrochaetae at the dorso/ventral boundary and in boundary cells between veins and interveins depends on Notch activity. We propose that at least during vein differentiation and wing margin formation, extramacrochaetae is regulated by Notch and collaborates with other Notch-downstream genes such as Enhancer of split-m(beta).Peer reviewe