Meeting Abstract
A next generation sequencing approach was employed to study global gene expression patterns in three distinct stages of limb regeneration, and in blastemas with disrupted and intact ecdysteroid signaling. SOAPdenovo-Trans assembly generated 208,404 contigs, averaging 583 nt. This database is available at http://www.genome.ou.edu/crab_Illumina.html and is both sequence and keyword searchable. This database was used to further explore the putative role of ecdysteroid signaling during the regeneration process. As proof of principle, the depth of sequence analysis has led to discovery of alternate EcR and E75 A/B domain isoforms, not identified through previous genomic or cDNA library screenings. EcR RNAi knockdown leads to a block in blastemal cell proliferation. Relative expression levels of cell division candidate genes between receptor knockdown and control libraries indicate a down-regulation of proliferation marker genes like PCNA, MCM2 and the cell cycle regulatory gene CycB in the knockdown library. This is accompanied by up-regulation of the nuclear receptor HR3, which inhibits CycB expression in insect systems. Excessive cuticle deposition seen in RNAi-treated animals correlated with an up-regulation of cuticular protein transcripts in knockdown libraries. Interestingly, cuticular proteins normally expressed at later molt cycle stages are aberrantly expressed in the early blastema. A lowering of circulating ecdysteroid titers observed as a consequence of receptor knockdown also correlates with an increase in MIH mRNA steady state transcripts in blastemal tissue of experimental animals. We postulate that an increase in MIH expression might in turn feedback systemically to lower circulating ecdysteroid titers below a critical level needed to maintain cell proliferation.
