Etude fonctionnelle de la Ténectine, un nouveau ligand des intégrines chez Drosophila melanogaster

Les intégrines sont des récepteurs de surface cellulaire permettant de relier le cytosquelette à la matrice extracellulaire présents chez tous les animaux et impliqués dans des processus clés au cours du développement comme la prolifération, la migration, la différenciation et la mort cellulaire. ténectine (tnc) est un nouveau gène codant potentiellement un ligand des intégrines alphaPS2betaPS chez la drosophile. Au cours de l embryogenèse, tnc s exprime dans de nombreux tissus d origine neuro-ectodermique comme les intestins antérieur et postérieur, les trachées et le système nerveux central. Afin de déterminer son rôle, nous avons généré des mutants par les techniques d interférence ARN et de remobilisation d un élément P. L analyse de leurs phénotypes et les résultats d expériences biochimiques et génétiques ont démontré que Tnc est un nouveau ligand des intégrines alphaPS2betaPS, impliqué dans l adhésion des épithélia alaires, la fasciculation axonal et la morphogenèse synaptique. Les résultats préliminaires suggèrent également que Tnc jouerait un rôle indépendant des intégrines dans la morphogenèse de l intestin postérieur et des trachées. Par ailleurs, plusieurs données nous indiquent que la 20 hydroxyecdysone et l hormone juvénile pourraient intervenir dans la régulation de l expression de tnc.Integrins are cell surface receptors of the extracellular matrix present in all animals and involved in key developmental processes such as cell migration, proliferation, differentiation and survival. tenectin (tnc) is a novel gene encoding a putative ligand of Drosophila alphaPS2betaPS integrin. In situ hybridization experiments revealed that tnc is expressed in many neuroectodermic tissues such as foregut, hingut, tracheas and central nervous system during embryonic development. In order to understand the role of tenectin during development we have established mutant lines, using RNA interference (RNAi) and P element remobilization processes. Phenotype analysis as well as biochemical and genetical experiments have demonstrated that Tenectin is a novel ligand of the Drosophila alphaPS2betaPS integrin and that Tnc is implied in wing epithelial attachment, axon fasciculation and synaptogenesis. Preliminary results suggest that tenectin plays although integrin-independant functions in trachea and hindgut morphogenesis. Moreover, some data suggest that tnc could be hormonally regulated by 20 hydroxyecdysone and juvenile hormone.DIJON-BU Sciences Economie (212312102) / SudocSudocFranceF

A novel putative insect chitinase with multiple catalytic domains: hormonal regulation during metamorphosis

0264-6021 (Print) Journal Article Research Support, Non-U.S. Gov'tWe have used differential display to identify genes that are regulated by juvenile hormone in the epidermis of the beetle Tenebrio molitor. One of the genes encodes T. molitor chitinase 5 (TmChit5), a chitinase possessing an unusual structure. Sequence analysis of TmChit5 identified five 'chitinase units' of approx. 480 amino acids with similarity to chitinase family 18. These units are separated by less conserved regions containing putative PEST (rich in proline, glutamic acid, serine and threonine) sequences, putative chitin-binding domains and mucin domains. Northern-blot analysis identified a single transcript of approx. 9 kb, whose abundance correlated with that of 20-hydroxyecdysone during metamorphosis. Injection of pupae with 20-hydroxyecdysone alone, or in combination with cycloheximide, indicated that TmChit5 expression is directly induced by the hormone. Further experiments indicated that methoprene (a juvenile hormone analogue) indirectly induced TmChit5 mRNA expression. On the basis of the present results and previous studies, we propose a molecular mechanism for cuticle digestion during the moulting process

Characterization of a putative extracellular matrix protein from the beetle Tenebrio molitor: hormonal regulation during metamorphosis

0949-944X (Print) Journal Article Research Support, Non-U.S. Gov'tWe used differential display to isolate epidermis cDNAs corresponding to juvenile-hormone analog-regulated mRNA from the beetle Tenebrio molitor. One of them encodes a putative extracellular matrix (ECM) protein, named Tenebrin. Indeed, the deduced protein sequence contains ECM typical features like the presence of a signal peptide, internal repeats, a RGD tripeptide sequence motif known to bind integrins and von Willebrand factor type c domains involved in protein-protein interactions. Northern blot analysis reveals a single transcript of about 11 kb with an expression pattern correlated to 20-hydroxyecdysone fluctuations during metamorphosis. In vivo injections of exogenous 20-hydroxyecdysone alone or combined with cycloheximide show that Tenebrin expression is directly induced by this hormone. Methoprene (a juvenile hormone analog) application experiments show that Tenebrin expression is rapidly induced by this analog. This gene is still up-regulated in the presence of protein synthesis inhibitor but, in these conditions, the mRNA induction level is not maximal

Developmental profiles of epidermal mRNAs during the pupal-adult molt of Tenebrio molitor and isolation of a cDNA clone encoding an adult cuticular protein: effects of a juvenile hormone analogue

0012-1606 (Print) Journal ArticleChanges in translatable mRNAs from the wing epidermis of the Coleoptera Tenebrio molitor have been investigated during metamorphosis by analysis of in vitro translated products. Striking differences between the patterns obtained from mRNAs extracted during pupal and adult cuticle secretion indicated that a drastic change in gene expression occurs during the pupal-adult transition. In addition to these stage-specific modifications, the mRNA patterns changed within each cuticular synthesis program (pupal or adult), especially at ecdysis. After tritiated leucine incorporation, some of the major radiolabeled cuticular proteins showed similar changes suggesting that the sequential appearance of mRNAs corresponds to sequential deposition of cuticular proteins. In supernumerary pupae obtained after juvenile hormone analogue (JHA) application on newly ecdysed pupae, translatable mRNA were very similar to those of pharate pupae. The JHA seemed, therefore, to prevent the expression of the adult program. By immunoblotting in vitro translated products with a monoclonal antibody recognizing an adult-specific cuticular protein, the developmental profile of the corresponding mRNA was studied. This mRNA was detected in anterior wing epidermis during the first 80 hr of the pharate adult stage. Using the same antibody, a cDNA clone was isolated from epidermal mRNA. The hybrid selected mRNA coded for only one protein with an apparent MW of 22 kDa which was, furthermore, recognized by the antibody. The Northern blot analysis performed with the clone confirmed the Western blot analysis of the in vitro translation products. JHA application at the beginning of the pupal-adult reprograming prevented the appearance of this mRNA; however, this transcript was present during the following molting cycle. This reversibility of the JHA action was confirmed by immunogold labeling of the cuticles formed in treated animals

Tenectin, a novel extracellular matrix protein expressed during Drosophila melanogaster embryonic development

1567-133X (Print) Journal Article Research Support, Non-U.S. Gov'tDuring Drosophila embryonic development, various morphogenetic processes require the remodeling of the extracellular matrix. In a previous study, we have identified and characterized a cDNA encoding a novel putative extracellular matrix protein named tenebrin, in the beetle Tenebrio molitor. Here, we examine the expression of the Drosophila ortholog, referred to as Tenectin (Tnc), during embryonic development. Tnc is expressed in the majority of tissues of neuroectodermic origin such as hindgut, foregut, tracheal system, anal plate, and CNS. In the CNS, the Tnc transcript is restricted to a few cells, whereas the protein is located in the dorsal part of the axonal tracts. In the hindgut and the trachea, Tnc protein is expressed on the apical pole of the cells. Tnc is an extracellular matrix protein secreted in a polarized way in different organs of Drosophila embryos

Cuticular protein genes in Tenebrio molitor (Coleoptera: Tenebrionidae)

1210-5759We have previously isolated from the beetle Tenebrio molitor, cDNAs coding for two glycine-rich cuticular proteins named ACP-20, ACP-22 and ACP-17 and an alanin-rich cuticular protein named LPCP-22. The ACP-20, ACP-22, ACP-17 mRNAs are detected by Northern blot and In situ hybridization analysis only in epidermal regions secreting heavily sclerotized cuticle during the pharate adult stage.The LPCP-22 mRNA is detected in most epidermal regions during the secretion of larval and pupal cuticles. Then, its presence is restricted to the epidermal zones secreting intersegmental soft cuticle in the newly ecdysed pupa. The stage- and tissue-specific gene system seems to be convenient model for studying the regulation of sequential gene expression by ecdysteroids and juvenile hormone

Characterization of a cDNA clone encoding a glycine-rich cuticular protein of Tenebrio molitor: developmental expression and effect of a juvenile hormone analogue

0962-1075 (Print) Journal ArticleThe complete sequence of a cDNA clone, isolated from epidermal mRNA of Tenebrio molitor using a monoclonal antibody raised against an adult-specific cuticular antigen only present in the hard cuticle, was obtained after primer extension at the 5' end. From this cDNA sequence, the deduced protein encompasses 199 amino acids (including a signal peptide) with a total molecular weight of 20.7 kDa. The protein exhibits a bipartite structure: glycine-rich region located in its NH2-terminal part and a carboxy-terminal domain sharing homologies with other cuticular proteins of Orthoptera, Diptera and Lepidoptera. In-situ hybridization analysis shows that the corresponding mRNA is present only in epidermal cells secreting the adult fibrous cuticle destined to become heavily sclerotized. In supernumerary pupae obtained after the application of the juvenile hormone analogue (JHA) to newly ecdysed pupae, the mRNA was undetectable, indicating that JHA can prevent the switch to the adult programme. However, in pupal-adult intermediates, obtained when JHA is applied later, the mRNA is detected

cDNA cloning and deduced amino acid sequence of a major, glycine-rich cuticular protein from the coleopteran Tenebrio molitor. Temporal and spatial distribution of the transcript during metamorphosis

0014-2956 (Print) Journal Article Research Support, Non-U.S. Gov'tIn Coleoptera, the elytra (forewings), with a very hard and thick cuticle, protect the membranous and delicate hindwings against mechanical stress. We have isolated and characterized a cDNA encoding a major cuticle protein in Tenebrio molitor, named ACP-20. The deduced amino acid sequence is roughly tripartite, with two terminal glycine-rich domains and a central region showing pronounced similarities with some other hard cuticle proteins. Northern blot and in situ hybridization analyses reveal that ACP-20 gene expression is developmentally regulated since transcript accumulation occurs only in epidermal regions synthesizing hard cuticle and is restricted to the period of preecdysial adult cuticle deposition. Moreover, application of a juvenile hormone analogue prevents the appearance of the transcript, indicating that juvenile hormone, a key molecule involved in the control of insect metamorphosis, negatively regulates the expression of the ACP-20 gene