Procedure
-Embryos:
-Fertilized chicken eggs were incubated in the laboratory at 37^o C with constant humidity until the desired stage (approximately 20 hours).

-Cyclopamine preparation and administration:
- 5 uL of Cyclopamine in 2-hydroxypropylcyclodextrin (HPCD) was administered to the embryo by injecting it through the vitelline membrane with a small pipette, through a small hole carefully cut in the shell.
- A solution of 45 % HPCD in PBS wasadministered in a similar fashion to all the control embryos.
- Embryos were allowed to continue developing in the incubator at 37 ^oC for an additional 48 hours.

- Staining Technique:
-Embryos were collected at 68 hours after initial incubation, or 48 hours after treatment. - Embryos were then washed with Howard's Ringer solution in a petri dish. Any extra- embryonic membranes were carefullyremoved with fine forceps.
- Embryos were covered with an appropriate volume of PI working solution (10 mg/ml PI in PBS) for approximately 10 minutes.
- Embryos were washed with Howard's Ringer and mounted on a depression slide. - Embryos were observed under a fluorescent microscope for the presence of dead cells in the tail bud and craniofacial regions.

- Physical Abnormalities:
-The embryos that were allowed to develop for a total of five days with cyclopamine were not stained and later observed under a dissecting microscope and photographed.