Introduction
Brandt Rakowski
Swarthmore College
Objective
This experiment attempted to determine the effects of the teratogen cyclopamine on the Shh signaling pathway in axolotl embryos. Groups of axolotl embryos that had undergone gastrulation were exposed to varying concentrations of ethanol or cyclopamine in ethanol. Their development was tracked over the subsequent 72 hours to determine if there were any congenital malformations as a result of exposure to the teratogens. Pictures were taken of the developing axolotls every 24 hours so their progress could be tracked.
Introduction
The Hedgehog proteins are a group of paracrine factors used by embryos to signal for the differentiation of cells and the creation of tissue boundaries during organogenesis. Of the three Hedgehog protein homologues held in common by all vertebrates, Sonic hedgehog (Shh) is responsible for the greatest number of functions. Produced by the notochord, it plays an important role in neural tube development, formation of somites, cranial-facial structure, left-right axis differentiation, tail bud formation, and organogenesis (Gilbert, 2003).
Cyclopamine, an alkaloid teratogen derived from the lily Veratrum californicum, is known to block the Shh signaling pathway in chick embryos by inhibiting cholesterol synthesis, which is needed for both Shh production and reception (Gilbert, 2003). Directly, cyclopamine inhibits Shh from binding to its protein receptors, rendering the signaling pathway useless (Gilbert, 2003). This results in a condition called holoprosencephaly, in which, depending upon the stage of embryonic development when the cyclopmanine is introduced, there is body truncation and various abnormalities in craniofacial structure, such as cyclopia in chicks, zebrafish, Medaka and Xenopus.
This experiment attempted to determine the effects of cyclopamine on axolotl salamander embryos that have undergone gastrulation. Embryo groups were exposed to various non-lethal levels of cyclopmanine suspended in ethanol, as well as equivalent levels of ethanol. The development of the embryos was qualitatively tracked for four days, with observations occurring every twenty-four hours. Because the embryos were introduced to the teratogens during a later stage when Shh signals for somite and tail bud differentiation, particularly close attention was given to the formation of lower body structure.