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.