Objectives Introduction Procedure Results Figure Discussion Acknowledgements and References

		Discussion Both the addition of ethanol to the solution and the addition of cyclopamine retarded the development of the embryos. The low-concentration solutions were retarded by the same amount, approximately 7-10 hpf, and development in the high-concentration solutions was inhibited even further. As development of the embryos in the low-concentration ethanol control was slowed by the same amount as that of the embryos in the cyclopamine solution, it cannot be concluded that the cyclopamine was responsible for this growth retardation at this concentration. The 60 mg/ml solution of cyclopamine does seem to additionally inhibit development beyond the retardation seen in the high-concentration ethanol control. The deformities in the ethanol control prevented accurate staging but the head appears to have developed further in that solution than in the cyclopamine solution. The cyclopamine embryos were staged based on head formation and yolk shape; thus while a sure conclusion cannot be drawn, it is believed that the cyclopamine at this concentration did impact the rate of development as the heads of the embryos in ethanol appeared further developed than those of the cyclopamine solutions. Different types of deformities were seen in the ethanol and the 20 mg/ml cyclopamine solutions, suggesting that even at this concentration, the cyclopamine affected embryo development beyond the effects of ethanol. The embryos in the ethanol solutions had deformities of the posterior end, the somites and the head while no posterior or somite deformities were seen in the embryos in the 20 mg/ml cyclopamine solution, which is unexpected because of the comparable amount of ethanol in the cyclopamine solution. The embryos in the cyclopamine solution did however have head deformities, as well as eye and circulatory deformities not seen in the ethanol solutions. The eye deformities such as the lack of eyes and the anteriorly positioned eyes were anticipated, since cyclopamine has been shown to cause cyclopia, as in the lambs (Gilbert 2003); and the head deformities could result from the undivided forebrain also responsible for the cyclopic condition. "Typical" cyclopia, the presence of one central eye, was not observed, however one embryo did have a single lens and no eyes, suggesting a similar mechanism, and reinforcing cyclopamine's role as an SHH inhibitor. The eyes observed in these embryos were underdeveloped for the stage of the embryo; most saliently they lacked the dark pigmentation of a normally developed eye. As SHH influences eye development in so many ways, perhaps this is another manifestation or side effect of its impairment. Further study would have to be undertaken to confirm that hypothesis. The circulatory deformities were unexpected and cannot be explained by past studies on cyclopamine or SHH function. The 60 mg/ml cyclopamine solution appeared to have severely retarded growth, although the embryos were too deformed to stage without uncertainty. The types of deformities observed were similar to those seen in the high-concentration ethanol solution, such as misshapen yolks, somite malformation, deformed posterior sections, and irregular heads. While some of these deformities are potentially attributable to cyclopamine, no conclusion can be drawn as they are also seen in the control. No deformities of the left-right axis were observed, although the heart, whose looping could indicate an L-R axis deformity, had not yet bent to either direction at the stages to which the cyclopamine treated embryos had developed. As the embryos were eaten (presumably) by the unicellular organisms in the solutions, no observation of them at an older stage was possible. The fact that the embryos in the cyclopamine solutions were eaten before those in the controls also suggests that the embryos were weaker than their control counterparts (although an alternative hypothesis is that the cyclopamine supported the unicellular organisms better than the controls, meaning that a greater number of them were present to attack the embryos), so development may not have continued much further anyways. Further experimentation must be undertaken to isolate the effects of cyclopamine on zebrafish embryos. The ethanol control results were anomalous with results observed in other ethanol solutions of similar concentrations, such as in those used in a similar experiment by Ferrari and Yanega (2000). The effects of both the ethanol controls and the cyclopamine solutions were much more extreme in this investigation than in theirs, which was performed on Medaka (Oryzias latipes) embryos. This could be due to a species specific impact of the concentrations used, suggesting that the faster development or another property of zebrafish embryos renders them more susceptible to teratogens than the slower developing saltwater Medaka. The impacts of ethanol on zebrafish should also be investigated thoroughly so that the malformations caused by ethanol and those caused by cyclopamine can be distinguished. Mindel (2000) noted a lack of eyes when zebrafish were treated with 2.5% ethanol solutions, suggesting that no conclusions, even those relating to eye deformities seen only in the cyclopamine treated embryos in this experiment, can definitively be drawn here.