Fig. 1

Adapting the Phytochrome System for Use in a Vertebrate Embryo

(A) 50-µm z projections through the hindbrains of 14-somite zebrafish embryos labeled with PHYB-MCherry-CAAX fusion proteins with different PHYB truncations. Dotted lines denote basal edges. N-PAS2-GAF-PHY-PAS contains a PAS domain at the C terminus and was not successfully expressed in six of six embryos. N-PAS2-GAF-PHY does not contain a PAS domain at the C terminus and was robustly expressed in cell membranes in nine of nine embryos.

(B) (i) Normal PHYB covalently binds PCB chromophore. Energy from far-red (FR) light causes photoisomerization of PCB and the allosteric transition of PHYB from its inactive (Pr) to its active (Pfr) state. This is reversible by infrared (IR) light exposure (not shown). The Pfr state can bind PIF. (ii) Conjugation of PCB with Y276H mutant PHYB creates an activated holoprotein that can directly bind PIF, without the need for far-red light illumination (Su and Lagarias, 2007). Energy from far-red illumination causes infrared fluorescence.

(C) An oblique confocal slice through the overlying EVL and the underlying neuroepithelium of a 14 hpf embryo, labeled with Y276H PHYB and H2A-GFP and bathed in PCB. Only the EVL fluoresces under far-red light (magenta), demonstrating binding of PCB to Y276H PHYB.

(D) A horizontal confocal slice through the neuroepithelium of an 18 hpf embryo, labeled with Y276H PHYB-CAAX and PIF6-EGFP and injected with PCB. Anterior is up. The membranes of all labeled cells fluoresced under far-red light, demonstrating binding of PCB to Y276H PHYB-CAAX. PIF6-EGFP was also recruited to the membrane in these cells. PIF6-EGFP recruitment to the membrane was not reversed after 5 min exposure to 750 nm light.

See also related Figure S1