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.

Procedure

1. Obtain several Hamburger & Hamilton stage 24 (d/d) axolotl embryos that have undergone gastrulation.

2. Remove jelly membrane using sharpened forceps.

3. Transfer dejellied embryos to 3 mL Holtfreter's solution.

4. Divide embryos into four experimental group Petri dishes and one control group Petri dish. Each dish should have a 3 mL Holtfreter's solution substrate. Label the dishes "control," "50 uM cyclopamine," "100 uM cyclopamine," "30 uL ethanol," and "60 uL ethanol."

5. Use cyclopamine at 2 mg/mL in ethanol as a stock. Dilute this solution in 10% HBSt to create two final solutions with concentrations 50 uM and 100 uM.

6. Add the 50 uM cyclopamine solution to the "50 uM cyclopamine" group, and add the 100 uM solution to the "100 uM cyclopamine" group.

7. Add 30 uL of ethanol to the "30 uL ethanol" group and add 60 of ethanol to the "60 uL ethanol" group.

8. Retain the control embryos in 3 mL Holtfreter's solution.

9. Keep embryos at 25° C for 24 hours.

10. Take still pictures and make observations.

11. Continue to make observations and take pictures every 24 hours until four observations total have been made.

Results

As expected, the control group continued to develop normally for the duration of the experiment (Fig. 1 & 2). The only malformations in this group were attributed to mechanical harm suffered during the de-jellying process, but this information will be discounted as insignificant as the damaged embryos continued to develop more or less normally.

The embryos exposed to both low (30 uL) and high (60uL) levels of ethanol were unable to survive longer than 48 hours. Prior to the fatal disassociations suffered by the embryos of these two experimental groups (Fig. 3), development seemed to appear normal.

The 50 uM cyclopamine experimental group also suffered 100% fatalities after 48 hours of exposure to the teratogen. This was, in part, due to mechanical trauma suffered during de-jellying. However, the undamaged embryos also exploded somewhere between H&H stages 33 and 37, most likely due to mechanical harm suffered during the de-jellying process. Prior to embryo death, slight malformations in the tail bud and body trunk region were detected in this experimental group (Fig. 4).

The most conclusive experimental evidence from this experiment can be observed in the 100 uM experimental group. Survival rate was 100% in this group, and development was tracked for a full 72 hours after exposure to the teratogens. Noted abnormalities include slight cranial malformations, including a smaller head and underdeveloped gill structures; a much shorter body trunk; and severe tail truncation (Fig. 5 & 6).

Figures

Figure 1. Control embryo at H&H stage 33.

Figure 2. Control embryo at H&H stage 45.

Figure 3. Deceased embryos from the 30 uL ethanol experimental group. Death is most likely attributed to mechanical stress during the de-jellying process.

Figure 4. 50 uM cyclopamine experimental group at H&H stage 33. Note slight truncation in body trunk and tail bud, as well as slight craniofacial abnormalities (compare to Fig.1).

Figure 5. 100 uM cyclopamine group at H&H stage 33. Note body truncation, especially in top subject.

Figure 6. 100 uM cyclopamine experimental group at H&H stage 45. Note underdeveloped gills and severe body and tail truncation (compare to Fig. 2).

Discussion

In this experiment, it was predicted that exposure to cyclopamine and ethanol would induce developmental malformations in the axolotl embryos, and that increased dosages of these teratogens would produce more severe defects. These predictions were made based on existing information that cyclopamine is known to block the Shh signaling pathway in some higher vertebrates (Gilbert, 2003).

The results of the experiment support the prediction that exposing embryos to cyclopamine causes defects in areas that depend on Shh during development. In particular, the 100 uM cyclopamine experimental group suffered severe body and tail truncation, which is closely associated with Shh inhibition. This sugests that axolotols share the same Shh signaling pathway with other higher vertebrates, such as birds and mammals, and establishes an evolutionary connection between these groups.

It was not predicted that the 50 uM cyclopamine group, as well as both ethanol experimental groups, would experience a 100% fatality rate. These results are especially confounding when the 100% survival rate of the 100 uM cyclopamine experimental group is taken into consideration. It is possible that the 100 uM group randomly consisted of hardier embryos, or that the 50 uM group and the ethanol groups randomly consisted of embryos that were more susceptible to teratogenic agents. It is also possible that most of the dead embryos suffered mechanical harm during the de-jellying process, which contributed to their early demise. However, this aspect of the experiment must remain inconclusive until further experiments using cyclopamine and ethanol on axolotls can be performed. Repetition of this experiment is the only way to be sure that random error did not play a role in skewing these data. It may also be worthwhile to expose axolotl embryos to amounts of ethanol less than 30 uL in order to determine the horizon of ethanol tolerance in this species.

Based on these data, it can be inferred that axolotls share a common signaling pathway with some birds and mammals. Does this mean that Shh exists in all vertebrates? Cyclopmine testing in a wider array of animals may help address this question. Also,is it possible to determine if evolution has consistently selected to conserve this trait in vertebrates? Answering these questions will help the scientific community gain greater insight into animal genetic history and the evolution.