By testing a series of patterns, we were able to find a patterning ‘sweet spot’ with strong rescue of many Nodal-dependent structures including somites, notochord, beating hearts and hatching gland.

To do this, we took advantage of the improved throughput of our microscope platform and created a ‘family’ of Nodal signaling patterns with different combinations of signaling intensity and pattern area.

We then used this platform to pattern Nodal-dependent developmental programs with targeted illumination. By directing light to the margin in Nodal loss-of-function mutants, we rescued endodermal precursor specification and internalization movements associated with gastrulation.

One technical trick that made this possible was 3D printing templates to mold embryo-sized ‘egg crate’ mounts. This allowed us make simple agarose devices to regularly array embryos for patterning in a format that allowed for easy retrieval afterward.

By leveraging ultra-widefield optics, we were able to spatially pattern Nodal signaling activity stimultaneously in 6 embryos per field of view. With automated scanning, we could increase this number to at least 36 embryos in one experiment.