Fig. 4.

EL222 optogenetic disruption of irf6 circumvents early embryonic lethality and causes a cleft palate phenotype. (A) Schematic of EL222 optogenetic system. VP16-EL222 monomers are inactive under dark conditions. Upon stimulation by 465 nm light, VP16-EL222 dimerizes, drives gene expression downstream of the C120 promoter and induces the expression of a dominant-negative form of irf6 (irf6-ENR). Embryos were exposed to blue light from 10 hpf to 72 hpf to circumvent embryonic lethality in mz-irf6^-8bp/-8bp embryos. (B-E) Brightfield microscopy of 72 hpf zebrafish embryos injected with the optogenetic system and grown in the dark (D) or exposed to blue light from 10-72 hpf (E) compared with control injected embryos (B,C). Injected fish exposed to blue light exhibit retrusion of the midface (arrowhead) and a curved body not observed in the other groups. (F-Q) Alcian Blue staining of cartilage and microdissection of the palate of 72 hpf embryos reveals a midface retrusion and cleft phenotype through the medial ethmoid plate (arrowhead in P, Q) in the C120-irf6-ENR-injected embryos grown under blue light (O-Q), which is not seen in control injected embryos (I-K) or injected embryos grown in the dark (L-N). Scale bars: 150 µm.