Cloning and characterization of the retinoid X receptor from a primitive crustacean Daphnia magna

Terpenoid hormones function as morphogens throughout the animal kingdom and many of these activities are mediated through members of the retinoid X group of nuclear receptors (RXR; NR2B). In the present study, RXR was cloned from the water flea Daphnia magna, a primitive crustacean of the class Branchiopoda, and characterized with respect to phylogeny, developmental expression, and hormonal regulation. The full length daphnid RXR cDNA was cloned by initial PCR amplification of a cDNA fragment from the highly conserved DNA-binding domain followed by extension of the fragment using RACE PCR. The full length cDNA was 1888 base pairs in length and coded for a 400 amino acid protein that exhibited the five-domain structure of a nuclear receptor superfamily member. The RXR protein shared significant identity with other NR2B group members. Phylogenetic analyses of the ligand-binding domain of the receptor revealed that daphnid RXR clustered with RXR from decapod crustaceans on a branch of the phylogenetic tree that was distinct from RXRs known to bind retinoic acids and juvenile hormones. Daphnid RXR mRNA levels were greatest in embryos that were early in development and progressively declined through the initial five stages of embryo development. Adult females expressed higher levels of RXR mRNA than did males and exposure of females to the terpenoid mimic pyriproxyfen reduced RXR mRNA to levels approaching levels in males. RXR mRNA levels in males were refractory to pyriproxyfen. These results show that branchiopod crustaceans dynamically express RXR which should be evaluated as a candidate receptor for the terpenoid hormone methyl farnesoate which functions as a sex determinant in these organisms.