Durica, D.S.*; Anilkumar, G.; Wu, X.; Hopkins, P.M.: A Search for EcR and RXR A/B Isoform Gene Expression During Crustacean Limb Regeneration and Oogenesis

We have previously reported the isolation of EcR and RXR gene homologs in the fiddler crab, Uca pugilator. In insects, isoforms for both the EcR and RXR/USP proteins have been identified; these isoforms arise from the splicing of alternative A/B domains onto common C and E/F domains. Expression studies and mutant analysis strongly suggest that these isoforms have different physiological roles. Detailed analyses of the recovered crustacean clones, however, recovered an invariant A/B domain open-reading frame for each gene. Ribonuclease protection assays (RPA) were employed to obtain an appraisal of the relative ratio of the recovered A/B domain-containing transcripts to common domain UpEcR and UpRXR sequences. RPAs were conducted using limb buds corresponding to limb regeneration stages used in previous studies and were also initiated with ovaries containing oocytes at different periods of oogenesis. To compare the relative steady-state abundance of A/B domain sequences to common domain sequence, RNAs were hybridized to both A/B domain and common domain probes, and the level of protection tritrated against known amounts of full-length sense cRNAs. At several stages examined, the relative level of A/B domain sequence protected is significantly less than common domain; additional bands of lower molecular weight resulting from hybridization to the A/B domain probe are also observed. Similar results were obtained using ovarian tissue samples. The difference in the abundance of A/B containing transcripts relative to total UpEcR and UpRXR mRNA suggests that alternative processing may produce A/B receptor isoforms in Uca, similar to the situation observed in insects. The ovary also represents another potential target tissue for ecdysteroid control.