Project description: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 â 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. Experiment Overall Design: 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.
Project description: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
Project description:Premetamorphic Xenopus laevis tadpoles limb bud cells respond to thyroid hormone by proliferation and subsequent differentiation. The goal of this experiment is to identify the genes involved in the TH-induced proliferation pathway in developing tadpole limb bud and compare it to TH-induced proliferation and differentiation program in tadpole brain. Xenopus tadpoles (NF54) were treated with 1 mM methimazole in 0.1 X MMR solution for 1 week to block the endogenous TH production and reduce the TH present in the system of the tadpole. They were then treated with 100 nM T3 in 1 mM methimazole and 0.1 x MMR for another 24h and 48h or without T3 for 48h (control group). Limb buds were dissected at the end of the experiment. Keywords: development or differentiation design,organism part comparison design,reference design,replicate design,time series design
Project description:Premetamorphic Xenopus laevis tadpole tail respond to thyroid hormone by resorption. The goal of this experiment is to identify the genes involved in the TH-induced resorption tadpole tail and compare it to TH-induced proliferation and differentiation program in tadpole limb and brain. Xenopus tadpoles (NF54) were treated with 100 nM T3 in 0.1 x MMR for another 24h and 48h or without T3 for 48h (control group). NF 61 tadpoles were in 0.1 X MMR till they reached NF stage 62. The tails were dissected after the experiment. Keywords: development or differentiation design,organism part comparison design,reference design,replicate design,time series design