Image acquisition and statistical analysis
Embryos and larvae were analyzed with Nikon SMZ 18 Fluorescence microscope and subsequently photographed with digital cameras. A subset of images was adjusted for levels, brightness, contrast, hue and saturation with Adobe Photoshop 7.0 software (Adobe, San Jose, California) to optimally visualize the expression patterns. Quantitative image analyses processed using image based morphometric analysis (NIS-Elements D4.6, Japan) and ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA; http://rsbweb.nih.gov/ij/). Inverted fluorescent images were used for processing. Positive signals were defined by particle number using ImageJ. 10 animals for each treatment were quantified and the total signal per animal was averaged. All data are presented as mean ± SEM. Statistical analysis and graphical representation of the data were performed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA). Statistical significance was performed using a Student’s t test , ANOVA, or χ2 test as appropriate. Statistical significance is indicated by *, where P < 0.05, and ***, where P < 0.0001.