Quantification and Normalization of Dorsal Gradient

Quantification of Dorsal gradient and normalization method are described in detail in [8] (link). Briefly, embryos were stained for anti-Dorsal antibody (Iowa Hybridoma Bank) and a Donkey anti-mouse Alexa 647, manually sliced in cross-sections within trunk region and imaged using a LSM700 Zeiss Confocal microscope. Fluorescent intensity from the 30-most ventral nuclei was obtained using Axiovision software (Zeiss). Position of midline was estimated with a double staining for snail RNA. For nuclei diameter measurement, early-stage embryos stained with anti-Laminin (Iowa Hybridoma Bank) were mounted longitudinally with glass beads (150–210 µm size, Polysciences), to prevent flattening caused by the coverslip. Confocal slices were taken from the embryo surface to its mid-section and nuclei diameter was determined using ImageJ software. In the case of ssm and gyn mutations, some additional measurements were taken from embryos stained with DAPI nuclear dye.