Su5402

Ripe adults of Branchiostoma lanceolatum were collected at the Racou beach near Argelès-sur-Mer, France (latitude 42° 32′ 53′′ N, longitude 3° 03′ 27′′ E). Gametes were obtained by heat stimulation as previously described (Fuentes et al., 2007 (link)) and staging is as described previously (Bertrand et al., 2021 (link); Carvalho et al., 2021 (link)). Before pharmacological treatment, embryos were transferred to scraped Petri dishes filled with filtered seawater to prevent the eggs from sticking to the plastic. InSolution SU5402 (572631; Sigma-Aldrich) at 10-2 M was added to cultures of embryos to a final concentration of 25 µM at the blastula stage [B stage, 5 h post-fertilization (hpf) at 19°C] and embryos were raised in this medium until fixation. Control embryos were raised simultaneously with equivalent concentrations of DMSO in filtered seawater. Embryos were fixed in paraformaldehyde (PFA) 4% in MOPS buffer, dehydrated in 70% ethanol and then kept at −20°C. Staging is according to Carvalho et al. (2021) (link).

The protocol for differentiation of pluripotent hiPSCs into RPE was adapted from Buchholz et al., 2013 [14 (link)]. For differentiation, iPSCs were plated at 70–80% confluence on 9.6cm² wells coated with 1X Matrigel (Corning) or Synthemax (3535XX1 Corning, Corning NY) or vitronectin (PeproTech) in basal neural induction medium (DMEM/F12+ 1X N2+ 1X B27 + 1X Non-essential amino acids, Thermo Fisher Scientific) supplemented for 2 days with Nicotinamide (10mM) (Sigma-Aldrich, St. Louis, MO), Noggin (50ng/ml) (PeproTech), Dkk-1 (10ng/ml) (R&D Systems, Minneapolis, MN.) and IGF-1 (10ng/ml) (R&D Systems) with a medium change on day 1. On days 3 and day 4, the Noggin concentration was reduced to 10ng/ml and bFGF was added at 5ng/ml (R&D Systems). The Nicotinamide, bFGF and Noggin were removed from the media on day 5 and on days 5 and 6 the basal medium was supplemented with only Dkk-1 (10ng/ml), IGF1 (10ng/ml) and Activin A (100ng/ml) (PeproTech). The DKK and IGF 1 were then removed and from day 7 to day 14 the basal medium was supplemented with Activin A (100ng/ml), and SU5402 (10μm) (Sigma-Aldrich). CHIR 99021(3μM) (TOCRIS, Bristol, UK.) was also added from day 8 to day14. (See Fig 1). All media supplements were removed for the expansion of iPSC-derived RPE.

The following chemicals were purchased from Sigma and used at the indicated concentrations and pre-treatment periods: Genistein (100 μM) for 10min, Vanadate (1 mM) for 30 min, PP1 (10 μM) for 5 min, PP2 (25 μM) for 5 min, K252a (100 nM) for 5 min, SU5402 (40 μM) for 60 min. Herbimycin A (5 μM) for 15 min and Lavendustin A (50 μM) for 15 min were purchased from Tocris. Bisindolylmaleimide (200 nM) for 30 min, was purchased from EMD. Forskolin (100 μM) was purchased from Sigma and Sf9 cells were treated with it for 6 min. The antigen and purification strategy for the anti-PKC Apl II antibody was previously described [22 (link)].

To prevent contractility for 2 hr, 100 hpf larvae were treated with 20 mM BDM (Higuchi and Takemori, 1989 (link)). To block Fgf and Hh signalling, zebrafish larvae were respectively exposed to SU5402 (15 µM) and Cyclopamine (5 and 10 µM) (Sigma-Aldrich) from 75 to 96 hpf. To increase retinoic acid signaling, dechorionated embryos were treated with 0.5 and 0.75 µM all-trans-retinoic acid (Sigma-Aldrich) from 74 to 96 hpf.