Meeting Abstract
P3.28 Thursday, Jan. 6 Transcriptome profiling of limb regeneration in the fiddler crab, Uca pugilator. DAS, S.*; NAJAR, F.Z.; HOPKINS, P.M.; ROE, B.A.; DURICA, D.S.; University ok Oklahoma sunetra.das-1@ou.edu
The crustacean Uca pugilator has the ability to regenerate its limbs as an adult. There are two phases of limb regeneration, basal and proecdysial growth, which normally correlate with molt cycle progression and variation in ecdysteroid titers and types. The basal growth phase is characterized by blastema formation and differentiation into a segmented ‘mini limb’. The proecdysial phase is marked by hypertrophic growth of the limb bud. Previous EST sequence data (via Sanger sequencing) suggests the existence of variable transcriptome profiles of the basal and proecdysial growth phases; however, genes and gene networks regulated by ecdysteroids during these growth phases are unknown. Here we begin characterization of the late proecdysial growth phase transcriptome via Roche 454 sequencing. To generate cDNA, we isolated RNA from late proecdysial limb buds (D1– D4 stage of molt cycle) from crabs with comparable blood titers, limb bud growth rate and size. 5µg of cDNA was generated in two steps using an amplification protocol that enriches for full length cDNAs and minimizes homopolymer error. During protocol optimization, pyrosequencing of this cDNA pool produced 554220 reads with an average read length of 248 nucleotides. These reads in turn were assembled into 4664 contigs and 52339 singletons. We are currently analyzing the compiled EST data to identify the metabolic pathways relevant to this regenerating phase. Analyses of the sequence data are available online at ftp://ftp.genome.ou.edu/pub/for_Durica/. Further transcriptome sequencing of both early blastema and blastemal limb regenerates from crabs with disrupted ecdysteroid signaling will be performed to identify gene and gene networks that are regulated by ecdysteroids and examine correlations to insect regeneration pathways.
