Nis elements advanced research image acquisition and analysis system

All images were obtained at 72 hpf when the live embryos were washed with 1×E3 and mounted in 3% methylcellulose. Whole body images were obtained at 10× on Nikon 80i compound microscope (Nikon Instruments). High magnification images of somites 11-13 were obtained at 20× using confocal microscope (Nikon A1R Si Confocal Eclipse Ti series). All confocal and software settings were kept unchanged during image acquisition. Images were processed with an NIS-Elements advanced research image acquisition and analysis system (Nikon Instruments), using the maximum intensity projection feature applied to z-stacks for greater depth of clarity. Image files were exported as TIFF files. Figures were composed with Adobe Photoshop CS6 (Adobe, USA).

To analyze the craniofacial skeleton, Alcian blue staining was performed on five WT and five β3^−/− embryos euthanized at 4.5 dpf, as described previously.³¹ Embryos were mounted in 95% glycerol in 1x phosphate buffered saline with tween 20 (PBST) and images were obtained at 10x and 40x on a Nikon 80i compound microscope (Nikon Instruments Inc., Melville, NY, USA). Images were processed with an NIS-Elements advanced research image acquisition and analysis system (Nikon Instruments), using the maximum intensity projection feature applied to z-stacks. Images for each animal were examined for abnormalities and measurements recorded for palate length and width as well as lower jaw length and width.