Transcriptomic Analysis of the Effects of Cyclopamine Exposure on Xenopus Limb Development

Study of the tetrapod limb has contributed a great deal to our understanding of developmental pathways and how changes to these pathways affect morphology. Most data on tetrapod limb development is known from amniotes, with far less known about genetic mechanisms of limb development in amphibians. To better understand the mechanisms of limb development in anuran amphibians, we use cyclopamine to inhibit Hedgehog signaling at various stages of limb development in Xenopus. We use transcriptomic analysis following cyclopamine exposure to understand the downstream effects of Hedgehog inhibition on gene expression. We find many aspects of Hedgehog function appear to be conserved with respect to amniotes, including the responses of ptc genes, gremlin, bmp2, and the autoregulatory property of shh. We show that, as was proposed based on experiments in chick, Sonic hedgehog plays two distinct roles in limb development – specification of digit number and specification of digit identity. In contrast to these points of conservation, we find that Hedgehog signaling is required for the maintenance of early limb bud outgrowth in Xenopus, a requirement not known for any other tetrapod. Keywords: pharmacological signal inhibition response Eight microarray experiments were performed using RNA extracted from batches of hindlimbs dissected from Xenopus laevis tadpoles. Four of these were experimental groups exposed to cyclopamine at 1 µg/ml for 24 hours before animal fixation, and four were experimental groups exposed to the ethanol, the cyclopamine vehicle, at the same concentration used for cyclopamine vehicle for 24 hours. Each pool of RNA consisted of material from 18-22 hindlimb buds. Two experimental groups and two control groups were from one clutch of tadpoles, and the other four pools were from another clutch. Both samples within each of four paired groups (experimental vs. control) were exposed and processed simultaneously.