Retinoids and steroids are known to modulate gene expression via binding to nuclear receptors (NRs), proteins encoded by a highly conserved family of genes that act as specific transcription factors in the presence of the signaling ligand. One focus of our laboratory has been an examination of the organization and evolution of gene families, and the programmed activation of evolutionarily related genes during invertebrate development. We have isolated genomic and cDNA clones from the fiddler crab representing homologs of the ecdysteroid (UpEcR) and retinoid-X (UpRXR) classes of NR, which are known to form the functional ecdysteroid receptor in arthropods. We have shown that, through differential mRNA processing, different structural variants (isoforms) of both receptors are produced during the molt and regeneration cycle. Interestingly, this variation occurs in the ligand binding domains of these heterodimer partners. We are also characterizing other crustacean nuclear receptors, whose homologs in insect systems work downstream of the immediate ecdysteroid signal.
Immunochemical localization of RXR
The amount and type of receptor that may be present in a given cell is too small to measure directly. Recombinant DNA techniques, however, allow development of molecular probes that can mark the presence of specific receptors at the cellular level, and microbes can be engineered to produce large amounts of receptor protein for immunological detection and physical studies. We can therefore detect which cells contain receptors, and by inference, identify particular cells that may be responding to steroid/retinoid signaling. The recombinant proteins can also be used to examine the physical characteristics of the receptors, such as their ability to bind to specific signaling molecules, the DNA sequences of potential target genes, and how changes in the protein sequence (either through differential processing or mutation) affect three dimensional architecture and protein-protein interactions.
Immunochemical localization of EcR
Several features of this system make it an attractive model for examining the role of NRs in the programming of the hormonal control of growth and development. Unlike most insects, crustaceans have a more complex adulthood, where the process of regeneration occurs in parallel with growth and reproduction. How a “common” endocrine modulator might regulate these varied activities is unclear. Although classical genetic analysis in this system is not possible, we have recently shown that we are able to perturb signaling through RNA interference.
Transcriptome analysis depicting specific differentially expressed genes (in green) between early blastemal development (4 days after limb loss) and early proecdysial limb bud development
The amenability of this system for monitoring hormonal changes is well established, and we now have the tools to identify potential hormonal target genes at the cellular level. We are now using transcriptomic tools to investigate changes in gene expression during regeneration over the molt cycle to examine the roles of ecdysteroid and terpenoid signaling.
Dr. Anilkumar Gopinathan, a Senior Professor at VIT University, India, has been a collaborator on work on crustacean ecdysteroid receptor function for over a decade.
