Dev Genes Evol (2004) 214:115–121 DOI 10.1007/s00427-004-0389-1
ORIGINAL ARTICLE
Vronique Royer · Alban Hourdry · Stphane Fraichard · Herv Bouhin
Characterization of a putative extracellular matrix protein from the beetle Tenebrio molitor : hormonal regulation during metamorphosis Received: 23 October 2003 / Accepted: 27 January 2004 / Published online: 17 February 2004 Springer-Verlag 2004
Abstract We used differential display to isolate epidermis cDNAs corresponding to juvenile-hormone analogregulated 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 20hydroxyecdysone 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 upregulated in the presence of protein synthesis inhibitor but, in these conditions, the mRNA induction level is not maximal. Keywords Metamorphosis · Ecdysone · Juvenile hormone · Extra cellular matrix protein · Molt
Edited by P. Simpson This work was supported by C.N.R.S. and Conseil Rgional de Bourgogne fundings V. Royer · A. Hourdry · S. Fraichard · H. Bouhin ()) Unit Mixte de Recherche CNRS 5548, Dveloppement-Communication Chimique, Universit de Bourgogne, 6 Boulevard Gabriel, 21000 Dijon, France e-mail:
[email protected] Tel.: +33-3-80396302 Fax: +33-3-80396289
Introduction Holometabolous insect metamorphosis transforms a larva into an adult though a complex series of developmental events involving cell proliferation and differentiation, programmed cell death, cell shape changes and cell rearrangements. During the postembryonic development of holometabolous insects, each molt is induced by a pulse of a steroid hormone (20-hydroxyecdysone or 20E), while the nature of the molt is controlled by a sesquiterpenoid hormone, the juvenile hormone (JH). Larval molts occur in the presence of high titers of JH while the metamorphic molts (pupal and adult) need low levels of JH (Riddiford 1994). While the molecular mode of action of ecdysteroids begins to be well understood, since the receptor and numerous target genes have been characterized (Thummel 1997, for review), the mechanisms of action of JH remain almost unknown because only a few rapidly regulated genes have been characterized and the nature of the receptor remains controversial (Hiruma 2003; Riddiford et al. 2003, for reviews). We are studying the metamorphic development of the wing of the beetle in an effort to understand the basic mechanisms that underlie epithelial morphogenesis and its hormonal control. In Diptera and Lepidoptera the wings originate from small clusters of undifferentiated cells constituting the imaginal disks (Oberlander 1985). In Coleoptera, the same process occurs in some families while in others, like Tenebrionidae, the wings differentiate from larval epidermal cells (Tower 1903; Quennedey and Quennedey 1990, 1999). In both cases, the cytoskeleton and the extracellular matrix (ECM) are reorganized but few hormonally regulated genes involved in those events have been characterized. Such studies, mostly performed on Drosophila imaginal disks, have shown that this remarkable transformation is coordinated by pulses of 20E, and requires the function of transcription factors (D’Avino and Thummel 1998) as well as ECM proteins and their receptors (Fristrom et al. 1993; Brabant et al. 1996; D’Avino and Thummel 2000), proteases (Fessler et
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al. 1993), and components of the contractile cytoskeleton (Fristrom et al. 1993). Effector genes expressed in the pupal wing thus appear to be good markers to study how JH and 20E pathways interact to control morphogenesis during metamorphosis. In the beetle Tenebrio molitor, a dramatic switch in epidermal gene expression is observed after the pupaladult reprogramming and these changes are prevented by application of a juvenile hormone analog (JHA; Bouhin et al. 1992a). In order to find new target genes of the JH, the differential display technique (Liang and Pardee 1992) has been performed on pupal wing mRNAs of T. molitor. In this study, we report the identification and characterization of a cDNA encoding a new putative ECM protein, whose expression is regulated by both developmental hormones.
Materials and methods Animals and hormonal treatments Mealworms, T. molitor (Coleoptera: Tenebrionidae), were mass reared at 25C on chicken food containing 1% yeast. Staging of larvae and pupae was carried out by the determination of physiological criteria based on the observation of eye migration (Delbecque et al. 1978) or in hours after pupation. JHA treatment was performed by topical application of 1 g methoprene dissolved in acetone on 16-h-old pupae. Molting hormone treatments were performed by injecting 2 g 20E dissolved in 2 l physiological serum into 4-h-old pupae. Cycloheximide (2 g) was injected as described before (Royer et al. 2002). mRNA isolation Wing epidermal RNAs were extracted from pupae as previously described (Bouhin et al. 1992b). For each extraction, RNA integrity was checked by gel electrophoresis. Poly (A)-rich RNAs were purified by oligo-(dT) cellulose affinity (Celano et al. 1993). In order to remove contaminating chromosomal DNA, mRNAs (2 g) were incubated for 15 min at 37C with 10 units recombinant RNasin ribonuclease inhibitor (Promega), 10 units DNase I (Life Technologies) in Tris-HCl 10 mM pH 8.3, 50 mM KCl, and 1.5 mM MgCl2. The reaction was stopped by an incubation of 10 min at 65C. Differential display mRNA differential display (Liang and Pardee 1992) was performed essentially according to Liang et al. (1993). For all samples, the reactions were carried out in duplicate. PCR were performed with the cycling parameters as follow: 94C for 30 s, 42C for 2 min, 72C for 30 s for 30 cycles followed by 72C for 5 min. The amplified cDNAs were then separated on a 6% sequencing gel. AmpliTaq DNA polymerase was obtained from Perkin-Elmer (Norwalk, Conn.) and (a-35S)-dATP (1,250 Ci/mmol) from Amersham. Recovery and reamplification of cDNAs The DNA sequencing gels were dried and autoradiographed. After developing the film, cDNA bands of interest were cut off from the gel. The candidate cDNA was diffused out by incubating the gel slice in 150 l bi-distilled water (ddH2O) for 15 min at 95C in a capped microfuge tube and ethanol precipitated using glycogen as a
carrier. The cDNA was redissolved in 10 l dH2O and reamplified as described except that (a-32P)-dATP was added in the reaction. The amplified fragment was then used as a probe in northern blot experiments. Northern blot experiments and northern capture Since one positive band could contain more than one cDNA, northern blots were used for affinity capturing the cDNA of interest (Li et al. 1994). Poly A+ RNAs (4.5 g) or RNAs (15 g) were separated by formaldehyde-agarose (1.1%) gel electrophoresis, transferred onto a Hybond-N+ membrane (Amersham) and UVcross linked (2,000 J/cm2). Prehybridization and hybridization were performed at 65C in 6 SSC, 5 Denhardt, 0.5% SDS, 100 g/ml salmon sperm DNA, and 10% dextran sulfate. Hybridization was performed for 16 h with a radiolabeled cDNA probe. After hybridization, the membrane was washed three times in 2 SSC, 0.1% SDS at room temperature for 20 min and twice in 0.2 SSC, 0.1% SDS at 65C for 30 min, then autoradiographed. The cDNA hybridized to the differentially expressed mRNA was eluted by incubating the membrane slice in 150 l ddH2O for 5 min at 95C in a capped microfuge tube. cDNA was ethanol precipitated and reamplified as described earlier. The PCR fragments were purified by agarose gel electrophoresis and cloned into the pMOSBlue Tvector kit (Amersham). cDNA library screening A lambda Zap II pupal epidermis cDNA library was screened with radiolabeled cDNA probes. Full length cDNA sequence was obtained by rescreening the cDNA library with PCR probes corresponding to the extremities of the cDNA already cloned. Slot blot analysis Total RNAs were brought to 10 SSC and slotted onto nylon membranes with a suction manifold. Hybridization was performed as described for northern blot experiments. To quantify autoradiographic signals, autoradiograms were scanned with a Shimadzu CS9000 densitometer equipped with an integrated computer. The Mann-Whitney U-test was used in statistical treatment of the data; only p
