Organ culture

Animals used for this study were euthanized at embryonic E14.5. Embryos were dissected under sterile conditions to retain a portion of the maxillary tissue containing the secondary shelves and using an intact upper lip to stabilize the tissue in a U shape with the medial edge epithelium of each shelf in contact with the other. Palates were cultured using a modified grid method in serum free BGJb medium (12591–038 Life Technologies, Grand Island, NY) containing 1X Pen/Strep for 72 h in 5 % CO² and 95 % room air at 37 °C in a humidified environment on sterile stainless steel grids. Following culture, palates were fixed and processed for paraffin embedding. 5 μm sections were cut in the coronal plane and stained with H&E. Images were visualized using a Nikon E1000 microscope.