Expression data from regenerating axolotl forelimbs

Salamanders are capable of regenerating amputated limbs by generating a mass of lineage-restricted cells called a blastema. Blastemas only generate structures distal to their origin unless treated with retinoic acid (RA), which results in proximodistal (PD) limb duplications. Little is known about the transcriptional network that regulates PD duplication. In this study, we identified expression patterns that explain PD duplication including upregulation of proximal homeobox gene expression and silencing of distal-associated genes whereas limb truncation was associated with disrupted skeletal differentiation. Overall, mechanisms were identified that regulate RAR’s multifaceted roles in the salamander limb including regulation of skeletal patterning during epimorphic regeneration, skeletal tissue differentiation during regeneration, and homeostatic regeneration of intact limbs. We performed microarray gene expression analysis on forelimbs that will eventually regenerate normally (DMSO-treated), become PD duplicated (RA-treated), or become truncated (LE135-treated). Genes were identified as statistically significant if they passed a false discovery rate (FDR) < 0.05 determined after an ANOVA analysis (533 significant probe sets), and changed from control DMSO samples > 1.5 fold change (FC) in either treatment group (327 significantly changed probe sets). Juvenile axolotls 8.8cm total length (TL) (high = 10.1cm, low = 7.4cm) and 4.58 average snout to vent length (SVL) received forelimb amputations at the distal zeugopod. Between days 7-14 dpa, individually housed animals were dosed with RA, LE135, or DMSO (n = 16/treatment). Drugs were changed every other day. Blastemas containing as little stump tissue as possible were collected from all 48 animals at 14 dpa and single forelimbs from four separate individuals were pooled together to yield four independent biological replicate samples for each treatment group.