Bms614

RXR is referred to RXRα hereafter. The pSG5-based Gal-RARα LBD (Gal-RARα), Gal-RXR LBD (Gal-RXR), Gal-TIF2 NRID (Gal-TIF2), Gal-SMRT NRID (Gal-SMRT), Gal-NCoR NRID (Gal-NCoR), RARα LBD-VP16 (RARα-VP16), RXRΔAB-VP16 (RXR-VP16), RXRΔAB, RARαΔAB, RXR, and RARγ expression vectors, and the (17m)5x-βGlob-Luc and the (RARE)3x-tk-Luc reporter genes have been described [52 (link),53 (link),54 (link)]. CD3254, UVI3003, LG100754 (LG754), LE135, CD2665, BMS961, BMS614, and BMS493 were from Tocris. BMS948 was provided by Bristol-Myers Squibb (New York, NY, USA) and AGN192870 (AGN870) by Galderma (Lausanne, Switzerland). UVI3002 was a gift of Angel de Lera (University of Vigo, Vigo, Spain). Am580 and TTNPB were from Sigma France (Saint Quentin Fallavier, France).

To block apoptosis, embryos were incubated with either 200 or 400 µM of the pan-caspase inhibitor Z-VAD-FMK (Promega) from 14 to 31 hpf (Williams et al., 2000 (link)). The drug was washed out and embryos were either fixed or incubated in fish water until 3 dpf and then fixed.
To activate RA pathway activity, embryos were soaked in 25 nM AM580 (Sigma), a pan-retinoic acid receptor (RAR) agonist, beginning at 24 hpf for the times indicated in figure legends. To inhibit the pathway, embryos were soaked in either 15 µM BMS493 (Sigma), an RARα inverse agonist, or 15 µM BMS614 (Tocris), an RARα antagonist, for 14-36 h from the 12-14 somite stage, which is after initial dorsal-ventral patterning of the eye and brain. After drug treatments, embryos were washed twice with embryo medium and then incubated until fixation with 4% paraformaldehyde at 28, 40 or 60 hpf. For optical manipulation of RA activity in the retina, Tg[RARE:YFP]^id1 embryos were soaked in 5 nM 13-cisRA (Sigma; Xu et al., 2012 (link)) at 24 hpf for 1 h in the dark and then mounted in 1.2% low melting point agarose. Photoactivation was performed with a single pulse of UV light (360-375 nm) illuminating the eye for 30 s, using a Zeiss 510 NLO two-photon microscope with 5 mW of power. Embryos were fixed at 33 hpf in 4% paraformaldehyde and immunostained for GFP.