Cloning and characterization of new orphan nuclear receptors and their developmental profiles during Tenebrio metamorphosis

Abstract

Five PCR fragments corresponding to a part of the DNA-binding domain of different hormone nuclear receptors were isolated from Tenebrio molitor mRNAs. The sequence identity of three of them with known Drosophila nuclear receptors strongly suggests that they are the Tenebrio orthologs of seven-up, DHR3 and b -FTZ-F1, and thus named Tmsvp, TmHR3 and TmFTZ-F1. The full-length sequences of the other two were established. TmHR78 is either a new receptor of the DHR78 family or the same gene which has evolved rapidly, particularly in the E domain. TmGRF belongs to the GCNF1 family and its in vitro translated product binds to the extended half site TCAAGGTCA with high affinity. The periods of expression of the corresponding transcripts in epidermal cells during Tenebrio metamorphosis were analyzed as a function of 20-hydroxyecdysone titers measured in the hemolymph of the animals taken for RNA extraction. Comparison of the expression profiles of these nuclear receptors with those observed during Drosophila metamorphosis revealed similar temporal correlations as a function of ecdysteroid variations, which further supported the sequence identity data for TmSVP, TmHR3, TmFTZ-F1 and TmHR78.Keywords: ecdysone; GCNF1; insect; metamorphosis; nuclear receptors.The pioneering work on chromosomal puffing induced by ecdysteroid in Drosophila salivary gland cells [1] gave rise to the idea that the molting hormone ecdysone triggered a regulatory cascade of gene expression. During the last decade, this hypothesis has been confirmed by molecular studies identifying the ecdysone receptor (EcR) [2] and several ecdysone-induced orphan members of the nuclear hormone receptor superfamily in Drosophila [3], and by developmental studies showing that many of these puff gene products are expressed hierarchically at various times after ecdysone action [4±6]. Such studies also suggest that in different tissues, the combinatorial expression cascade of these ecdysone-induced receptors may be different, thus contributing to the specificity of response to the hormone.The identification of several orthologs of Drosophila nuclear hormone receptors in other insect species supports the idea that the regulatory cascade found in the fruit fly could be conserved throughout this class. However, differences occur at the structural and transcriptional levels among different insect species studied so far [3], which could explain the differences observed in their life-cycles. Indeed, even in the apparently homogenous group of holometabolous insects, the metamorphic events present such striking differences in timing and cell fate among different orders that it is of interest to compare the cascade of molecular events triggered by ecdysone described for Drosophila with that of other metamorphosing insects. The identification of Drosophila EcR homologs in several other holometabolous species [7±15] supports this molecular diversity hypothesis. However, less is known concerning the homologs of other nuclear receptors characterized in Drosophila and thus it seems in...

Origins and evolutionary diversification of the nuclear receptor superfamily

“…Similarly, Garcia-Vallvé and Palau [83], using different algorithms, have placed the TR and RAR into separate sub-families than Laudet [50]. The arthropod orphan receptors THR4 [102] and GRF [Charles et al, unpublished accession AAD38900] cluster in phylogenetic trees with GCNF, which until recently was regarded as the sole member of sub-family VI (see fig. 4).…”

Functions of Nuclear Receptors in Insect Development

“…HR4 gene is directly induced by 20E in all insect species analyzed [70,71,72]. Remarkably, its transcript always appears at the same time or just a little after HR3 and before FTZ-F1, suggesting that HR4 is another important member of the 20E-dependent genetic network (Figure 3.2) [73,74,70,75,18]. …”

“…Although HR78 expression is induced by 20E in cultured organs and its mRNA levels oscillate with each molting cycle [100,74,18], several results show that it is a negative modulator of EcR-USP/RXR action. First, in D. melanogaster, DHR78 binds to a subset of EcR/USP binding sites [100].…”

“…Svp is a potent repressor that is widely expressed throughout development [116,117,74,118]. In D. melanogaster and A. aegypti, SVP represses the 20E-dependent transactivation mediated by EcR-USP [119,118].…”

Arthropod-Inducible Proteins: Broad Spectrum Defenses against Multiple Herbivores

“…CmCatB was the most highly induced gene in a microarray study designed to identify scNregulated genes in cowpea bruchid alimentary tracts. Chicken ovalbumin upstream promoter-transcription factors are orphan nuclear receptor family members, previously known to play important roles in neurogenesis, organogenesis, and embryogenesis in various animals (Kerber et al, 1998;Mouillet et al, 1999). In the bruchid midgut, this transcription factor acts as a repressor of CmCatB expression under normal growth conditions.…”