Fig. 2.

Platform for spatial and temporal patterning of Nodal signaling activity. (A) Schematic of patterning experiment. One-cell wild-type embryos were injected with mRNA encoding optoNodal2 receptors (15 pg per receptor). At sphere stage, embryos were mounted in custom array mounts compatible with an upright microscope. Spatial patterns of light were generated using an ultra-widefield microscope incorporating a DMD-based digital projector (Fig. S4). Patterns were applied with average intensity of 20 µW/mm². (B) Experimental timeline. Embryos were injected with optoNodal2 mRNAs (15 pg per receptor mRNA) at the one-cell stage. Embryos were kept in the dark until 4 hpf. Embryos stained for pSmad2 (D) were illuminated from 4-4.3 hpf, while embryos stained for lft2 or noto expression (E,F) were illuminated from 4-4.75 hpf (‘+hν' indicates illumination). All embryos were fixed immediately after light treatment. (C-F) Demonstration of spatial patterning of Nodal signaling activity and target gene expression. (C) DMD pattern masks used for spatial patterning. (D) α-pSmad2 immunostaining (green) demonstrating spatial patterning of signaling activity. (E) Spatial patterning of noto gene expression (cyan). (F) Spatial patterning of lft2 gene expression (yellow). Embryos were double stained for lft2 and noto; each column of images in E and F depict the same embryo imaged in different channels. All images in D-F are maximum intensity projections derived from confocal images of a representative embryo. Scale bars: 100 µm.