Immediately after sorting all animals into the appropriate tanks, 30μl per animal of 5mg/ml cyclopamine solution was added to all experimental tanks (to a final concentration of 1 μg/ml, and 30μl per animal of 100% ethanol was added to each control tank. Tanks were rocked gently to distribute cyclopamine and ethanol. Exposures were carried out for 24 hours and ended with fixation of treated animals.
Approximately 200 tadpoles from each of two clutches (designated “clutch A” and “clutch B”) of the species Xenopus laevis were obtained from Nasco Biology for a total of approximately 400 tadpoles. Animals arrived at approximately stage 45. All animals were kept in a room with an air temperature of 25ºC ± 1ºC. After one day of equilibrating to room temperature, animals were moved to an Aquatic Habitats aquatic housing unit (http://www.aquatichabitats.com/, Apopka, FL). Thirteen tadpoles were put into each of twenty-four tanks, without mixing clutches, for a total of 156 animals of each clutch. Each tank was filled to 9 liters with treated water. Tap water was treated and aged for at least one day with approximately 1ml Stress Coat (Aquarium Pharmaceuticals, Chalfont, PA) for every 22 liters of water. To decrease temperature-based variability in developmental rates between clutches due to slight temperature variations in the room, tanks were oriented on the rack in four rows of six tanks, with tanks along each row or column alternating between clutches. To decrease temperature-based variability in developmental rates within clutches, every three days, tanks on the top row were switched with those on the bottom row, and the middle two rows were similarly exchanged. All animals were staged each day, and animals at stage 52 (Nieuwkoop and Faber, 1994) were set aside for exposure. The population of animals at stage 52 from each clutch was divided in half indiscriminately, resulting in four groups: (1) a control group for clutch A, (2) an experimental group for clutch A, (3) a control group for clutch B, and (4) an experimental group for clutch B. These groups were then moved to new plastic exposure tanks. If a group was over twenty animals it was split among more than one exposure tank so that no tank had more than twenty animals. Each exposure tank was filled with 150ml treated water per tadpole. All exposure groups throughout the experiment had at least ten individuals, so exposure tank volumes ranged from 1,500ml (for ten individuals) to 3,000ml (for twenty individuals).
Both hindlimb buds were dissected off of each animal at the base of the limb using surgical scissors. For animals from exposure tanks with over eleven individuals, limbs were divided into sub-groups of between eighteen and twenty-two limbs. This resulted in six groups of limbs per clutch for control groups and six groups of limbs per clutch for experimental groups, or a total of twenty-four groups of limbs. All limbs were placed in fresh vials of RNAlater, and returned to -20ºC for continued storage. Twenty-four total RNA preparations were extracted, in several batches of between four and six preparations each batch, using the RNeasy Mini Kit and optional RNase-Free DNase Set (QIAGEN, Valencia, CA), with the following notes. Limbs were put into 1.5ml microcentrifuge tube, residual RNAlater was pipetted off. Limbs were crushed with a homegenizer in 200μl buffer RLT, then 300μl buffer RLT was added. Elution was carried out with two washes of 50μl RNase-free water. Extracted total RNA was stored at -80ºC and transferred to the W. M. Keck Foundation Biotechnology Resource Center, Affymetrix Resource Center (Yale University, New Haven, Connecticut). All samples were again run through DNase treatment. Eight of the twenty-four RNA extractions were chosen for hybridization.
Labeled according to standard Affymetrix protocols, performed by the Yale University W. M. Keck Foundation Biotechnology Resource Laboratory
Hybridized according to standard Affymetrix protocols, performed by the Yale University W. M. Keck Foundation Biotechnology Resource Laboratory
Scanned according to standard Affymetrix protocols, performed by the Yale University W. M. Keck Foundation Biotechnology Resource Laboratory
Cyclopamine Experimental 1 Normalized Expression Value (Clutch A, Group 1, M1)
Data were normalized by the Yale University W. M. Keck Foundation Biotechnology Resource Laboratory. GeneChip Operating Software was used to calculate the overall intensity of each arrray by averaging the intensity values of every probe set on the array with the exception of the top and bottom 2% of the probe set intensities. For each array, the average intensity of the array was multiplied by a scaling factor for that array to bring it to an arbitrary target intensity value common to all arrays.