Fig. 3.

Rescue of endoderm precursors and internalization movements. (A) Endoderm rescue experiment. In wild-type embryos, Nodal signaling near the margin turns on the master endoderm transcription factor sox32 at 4 hpf. By 6 hpf, sox32⁺ endodermal precursors have internalized, and by 9 hpf they have spread over the yolk via random walk movements. MZeop mutants lack Nodal signaling and do not specify endoderm. We rescued sox32 expression and downstream cell movements in MZoep embryos by targeted optoNodal2 stimulation at the margin from 3.75 to 6 hpf (indicated by blue bar). Embryos were injected with 30 pg of optoNodal2 mRNA. Illumination patterns had average powers of 40 µW/mm². (B) Rescue of sox32 expression at 6 hpf expression with optoNodal2 stimulation. sox32⁺ cells were visualized by hybridization chain reaction in wild type (top row), MZoep (middle row) and optoNodal2-stimulated MZoep embryos (bottom row). Insets and white arrow highlight localization of sox32⁺ cells at the embryonic margin. Asterisk highlights Nodal-independent sox32 expression in the extra-embryonic yolk syncytial layer. (C) Rescue of cell internalization movements with optoNodal2 stimulation. sox32⁺ cells were visualized by HCR at 9 hpf in wild type (top row), MZoep (middle row) and optoNodal2-stimulated MZoep (bottom row). Insets depict maximum intensity projections of middle confocal slices to visualize the hypoblast cell layer. sox32⁺ cells reside in the hypoblast in wild-type and optoNodal2-treated embryos at 9 hpf. Scale bars: 100 µm.