Meeting Abstract
92.6 Wednesday, Jan. 7 Expression of ecdysteroid responsive genes in response to hormonal induction and RNAi mediated gene silencing in Uca pugilator DAS, S.*; HOPKINS, P.M.; DURICA, D.S.; University of Oklahoma; University of Oklahoma; University of Oklahoma sunetra.das-1@ou.edu
The ecdysteroids, steroid hormones of arthropods, regulate growth, reproduction and limb regeneration in crustaceans by regulating gene expression. We have taken two different approaches to study ecdysteroid responsive genes in Uca pugilator. One approach involved examining EcR, the ecdysteroid receptor gene, and E75, a related nuclear receptor gene, whose transcriptional activity in insect systems increase as a direct result of hormone induction. To test whether endogenous crustacean ecdysteroids affect these candidate genes, newly hatched synchronously growing larvae were incubated in various concentrations of ecdysteroids (ecdysone, 20-hydroxy-ecdysone or ponasterone A) for 3 hours. RT-PCR results indicated a 2 to 5 fold increase in UpEcR (p=0.03) and UpE75 (p=0.001) transcript abundance when incubated with ponasterone A, but not with the other two hormones, indicating that these two genes are primary response genes to ponasterone A. A second approach involved RNAi mediated knockdown of either UpEcR, or its heterodimer partner in the functional ecdysteroid receptor complex, UpRXR, during limb regeneration. For these experiments, RNAi injections were performed on day 1 and 4 following autotomy (limb loss), where a 207nl volume of dsRNA (420-840ng) was applied directly below the autotomy membrane in proximity to the developing blastema. RT-PCR results showed that injection of dsRXR leads to a 2 fold decrease of UpRXR transcripts in day 6 blastemas (p=0.04); dsEcR injection, however, did not show a significant decrease, with high variability between animals. To standardize blastemal samplings, injected limb buds from crabs with comparable levels of circulating ecdysteroids are currently being tested for receptor knockdown, and changes in growth rate and morphology.
