Hervé Bouhin

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

Changes 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.

Molecular cloning and expression of Tenebrio molitor ultraspiracle during metamorphosis and in vivo induction of its phosphorylation by 20-hydroxyecdysone

Using a RT‐PCR approach, the Tenebrio molitor homologue of Drosophila Ultraspiracle (TmUSP) was characterized. Its DNA binding domain shows a degree of identity with those of the other insect USPs. However, the ligand binding domain is closer to those of retinoid X receptors. Using an antibody raised against DmUSP, Western blot analysis of proteins from epidermis and other tissues revealed five immunoreactive bands, corresponding to different phosphorylated forms of a unique polypeptide, as shown by λ‐phosphatase treatment. The nuclear form of TmUSP seems unphosphorylated. An in vivo 20‐hydroxyecdysone treatment increases considerably and rapidly the phosphorylated forms of TmUSP. This post‐translational modification may play a role in the 20‐hydroxyecdysone response.

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

The 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.

Characterization of two new cuticular genes specifically expressed during the post-ecdysial molting period in Tenebrio molitor

In a previous study, we have isolated a cDNA, TM-ACP17, coding for a post-ecdysial adult protein of Tenebrio molitor. After screening of a genomic library with TM-ACP17, we report isolation and sequencing of TM-ACP17 gene and a new gene, TM-LPCP29, coding for a larval-pupal protein. These two genes exhibit a common sequence of 15 nucleotides and a characteristic of most cuticular protein genes so far described: an intron interrupting the signal peptide. The deduced aa sequence of TM-LPCP29 exhibits a high percentage of Ala (26.5%) and Val (17.5%) and is highly hydrophobic. In the N-terminal part, the motif VAAPV is repeated ten times. Numerous histidine residues are present in the C- and N-terminal regions. A comparison is made with other cuticle protein sequences. Northern hybridization analysis showed that TM-LPCP29 is present during larval and mainly pupal post-ecdysial cuticle secretion. In-situ hybridization revealed that TM-LPCP29 mRNA is expressed in epidermis and not in muscles or fat body.

A luminal glycoprotein drives dose-dependent diameter expansion of the Drosophila melanogaster hindgut tube.

An important step in epithelial organ development is size maturation of the organ lumen to attain correct dimensions. Here we show that the regulated expression of Tenectin (Tnc) is critical to shape the Drosophila melanogaster hindgut tube. Tnc is a secreted protein that fills the embryonic hindgut lumen during tube diameter expansion. Inside the lumen, Tnc contributes to detectable O-Glycans and forms a dense striated matrix. Loss of tnc causes a narrow hindgut tube, while Tnc over-expression drives tube dilation in a dose-dependent manner. Cellular analyses show that luminal accumulation of Tnc causes an increase in inner and outer tube diameter, and cell flattening within the tube wall, similar to the effects of a hydrostatic pressure in other systems. When Tnc expression is induced only in cells at one side of the tube wall, Tnc fills the lumen and equally affects all cells at the lumen perimeter, arguing that Tnc acts non-cell-autonomously. Moreover, when Tnc expression is directed to a segment of a tube, its luminal accumulation is restricted to this segment and affects the surrounding cells to promote a corresponding local diameter expansion. These findings suggest that deposition of Tnc into the lumen might contribute to expansion of the lumen volume, and thereby to stretching of the tube wall. Consistent with such an idea, ectopic expression of Tnc in different developing epithelial tubes is sufficient to cause dilation, while epidermal Tnc expression has no effect on morphology. Together, the results show that epithelial tube diameter can be modelled by regulating the levels and pattern of expression of a single luminal glycoprotein.

Identification, sequence and mRNA expression pattern during metamorphosis of a cDNA encoding a glycine-rich cuticular protein in Tenebrio molitor

The study of insect cuticular proteins and their sequences is of interest because they are involved in protein-protein and protein-chitin interactions which confer the mechanical properties and fine architecture of the cuticle. Moreover, in the coleopteran Tenebrio molitor there is a dramatic change in cuticular architecture between pre- and postecdysial secretion. We report the isolation, by differential screening, and the sequence characterization of a cDNA clone encoding a cuticular protein of T. molitor, ACP17. After insertion in the expression vector pEX1, the recognition of the fusion protein by an anti-cuticular monoclonal antibody confirmed the cuticular nature of ACP17. Northern hybridization analysis showed that ACP17 mRNA expression begins weakly 3 days before adult ecdysis and strongly increases during the secretion of postecdysial adult cuticle, with a maximum just after ecdysis. In situ hybridization revealed that the ACP17 mRNA is only present in the epidermis which secretes hard cuticle. The deduced amino acid (aa) composition exhibits a high content of Gly (28%) and Ala (20%) and, particularly, two poly(Gx) stretches separated by repetitive motifs with proline AAPVA. A comparison is made with other cuticle aa sequences.

Nucleotide sequence of an adult‐specific cuticular protein gene from the beetle Tenebrio molitor: effects of 20‐hydroxyecdysone on mRNA accumulation

The accumulation of transcripts from two adult‐specific cuticular genes (ACP‐20 and ACP‐22) is shown to be modified after addition of exogenous 20‐hydroxyecdysone. In the continuous presence of high levels of the hormone, the expression of ACP‐20 gene is significantly weaker than that of untreated controls, while ACP‐22 expression is 2.5‐fold increased. During active synthesis of the ACP messages, a 0.5 µ20‐hydroxyecdysone injection causes a rapid 2‐fold increase in ACP‐22 mRNA and is not able to repress ACP‐20 mRNA accumulation. We conclude that these genes whose transcripts appear in an almost coordinated manner in epidermal cells during the moulting cycle are regulated by ecdysteroids in a different way. In order to undertake a functional dissection of the promoter regions of ACP‐22 gene, we have isolated and sequenced a genomic clone. The sequence similarities with other cuticular protein genes are discussed.

Tenectin is a novel αPS2βPS integrin ligand required for wing morphogenesis and male genital looping in Drosophila

Morphogenesis of the adult structures of holometabolous insects is regulated by ecdysteroids and juvenile hormones and involves cell-cell interactions mediated in part by the cell surface integrin receptors and their extracellular matrix (ECM) ligands. These adhesion molecules and their regulation by hormones are not well characterized. We describe the gene structure of a newly described ECM molecule, tenectin, and demonstrate that it is a hormonally regulated ECM protein required for proper morphogenesis of the adult wing and male genitalia. Tenectins function as a new ligand of the PS2 integrins is demonstrated by both genetic interactions in the fly and by cell spreading and cell adhesion assays in cultured cells. Its interaction with the PS2 integrins is dependent on RGD and RGD-like motifs. Tenectins function in looping morphogenesis in the development of the male genitalia led to experiments that demonstrate a role for PS integrins in the execution of left-right asymmetry.

Changes of in vitro translation products from the RNA in the posterior caeca of the crustacean Orchestia during the molt cycle

Abstract 1. 1. Changes in the translatable mRNAs from the midgut posterior caeca of the crustacean Orchestia were investigated during the successive molting stages by analysis of the products of in vitro translation: the changing banding pattern of the in vitro synthesized polypeptides indicated changes in the cellular pattern of mRNAs. 2. 2. Only some weak differences were observed in intermolt between cell-type I (in the distal segment) and type II (in the proximal segment). Major changes appeared during molting, with the change of expression of epithelial cells, through striking quantitative differences between cell-type III (in preexuvial period) and type IV (in postexuvial period). 3. 3. These biochemical developmental changes are discussed in connection with the developmental changes of the ultrastructural characteristics of epithelial cells previously investigated.