Fig. 4.

Optogenetic rescue of Nodal signaling mutant phenotypes. (A) Experimental overview. The absence of Nodal signaling in MZoep mutants (middle) results in loss of nearly all mesendodermal tissues. We injected optoNodal2 mRNA (15 pg/receptor) into one-cell stage MZoep embryos and replaced endogenous Nodal signaling with patterned optogenetic stimulation (bottom). (B) Experimental timeline. Illumination patterns were applied from 3.5 to 6 hpf with the indicated powers. Embryos were imaged or fixed at 26 hpf. (C) Schematic of arrayed layout of Nodal patterns. Optogenetic pattern characteristics were varied along each axis of the embryo array; pattern geometry was varied left-to-right, and pattern intensity was varied top-to-bottom. (D) Visualization of stimulation patterns. Applied patterns (green) were visualized by projecting pattern masks with 560 nm illumination and observing fluorescence from a co-injected mCherry mRNA. Each combination of pattern geometry and intensity was tested in five or six replicate embryos in the depicted experiment. The 26 hpf phenotypes of boxed embryos are highlighted in C. (E) Example rescue phenotypes. Example of a partial rescue phenotype (top), exhibiting notochord, trunk somites and partial rescue of cyclopia. Weaker intensity stimulation (middle) resulted in weaker rescue, with incomplete specification of trunk somites and notochord. Combining high intensity and large-area stimulation led to phenotypes reminiscent of Nodal gain-of-function (bottom, e.g. lefty1;lefty2 double mutants). Scale bar: 100 µm. (F) Quantification of rescue phenotype frequencies for trunk somites (left), notochord (middle) and embryo disruption due to hyperactive Nodal signaling (right). Phenotypes were assessed by visual inspection of transmitted light images. (G) Visualization of marker gene expression for Nodal-dependent tissues. Top row: expression of notochord (shha, green) and hatching gland (ctslb, yellow) markers. Bottom row: expression of somite (myod1, green) and hatching gland (ctslb, yellow) markers. Scale bar: 100 µm.