Objectives Introduction Procedure Results Figure Discussion Acknowledgements and References

		Materials Cyclopamine ( ) 2 mg/ml in ethanol 100% ethanol 60 mm glass pretri dishes Wide-mouth pasteur pipets and bulbs Incubator at 28°C Dissecting microscope with camera Pipetmen and tips Fine forceps Procedure 1. Harvest embryos. 2. Observe embryo development microscopically and stage. 3. Prepare five glass Petri dishes with 5.0 mL of the one of the solutions each. Label the dishes by recording the contents. The five solutions are as follows: * 150 µL cyclopamine (CY) in 5mL ZF embryo medium (high-concentration, 60 mg/ml CY) * 50 µL cyclopamine in 5mL ZF embryo medium (low-concentration, 20 mg/ml CY) * 150 µL ethanol in 5mL ZF embryo medium (high-concentration) * 50 µL ethanol in 5mL ZF embryo medium (low-concentration) * 5 mL ZF embryo medium 4. Divide the embryos between the five dishes once they reach 30% epiboly (about 4 hours 40 minutes after fertilization, although observation provides a surer approximation of staging). Transfer them with wide Pasteur pipets.. Gently puncture the chorion of each embryo once it has been transferred by tearing the chorion with fine forceps. Practice this before, and do not worry about removing the whole chorion, as this makes injury to the embryo more likely. 5. Incubate dishes at 28ºC. 6. Observe and photograph embryos after 24 hours. Note any deformities. Remove empty chorions.