Vectors and procedures used to expand TRAP approaches in zebrafish.(A): The Tol2_trap:TRAP vector comprises a cassette containing the eGFP-rpl10a fusion gene (green) and the gata2p minimal promoter (brown), flanked by Tol2 recognition sequences (orange). This vector was injected together with Tol2 transposase mRNA in one-cell stage zebrafish embryos. Once grown, adult fish were screened for eGFP-rpl10a expression in their progeny. (B): Pie charts showing the efficiency rate of the trap:TRAP approach. (C): The Tol2_UAS:TRAP vector comprises a cassette that contains the eGFP-rpl10a fusion gene (green) together with the 5xUAS element (blue), flanked by the Tol2 recognition sequences (orange). This vector together with Tol2 transposase mRNA were injected together in one-cell stage embryos. Once grown, adult fish were outcrossed with a Gal4 line to identify founders. (D): Schematic representation of the Tol2_vsx2.2:TRAP vector used to test the functionality of the eGFP-rpl10a cassette (green) under the control of the vsx2.2 promoter (red). (E): Retina-specific eGFP-rpl10a expression in the line Tg[vsx2.2:TRAP] tested in TRAP-seq experiments at 22 hpf. L, lens. Scale bar = 100 µm. (F): Differential expression (RPKM) of retinal TF-encoding genes in affinity-purified transcripts from vsx2.2:TRAP embryos at 22 hpf (TRAP-seq, n = 3, green), and in 22 hpf whole embryos RNA-seq sample (control, blue). Note the significant expression enrichment of neural retina-associated transcription factors (ranked by fold-change) in the vsx2.2:TRAP sample (p = 0.0004; two-tailed paired t-test). RPKM values corresponding to TFs from the core retinal GRN, as well as their fold change vs the control, are indicated.

eGFP-rpl10a expression patterns: (A,A′): TT1, hindbrain and spinal chord (lateral and dorsal, respectively); (B,B′): TT5, jaw, brachial arches and pectoral fin buds (lateral and ventral, respectively); (C): TT6, skeletal muscles (lateral); (D): TT7, central nervous system (lateral); (E): TT15, lateral line system (lateral); (F, F′): TT21, rhombomere 5 (lateral and dorsal, respectively); (G,G′): TT28, hindbrain and pectoral fin buds (lateral and dorsal, respectively); (H,H′): TT37, retina (lateral and ventral, respectively); (I,I′): TT42, midbrain stripe (lateral and dorsal, respectively); (J): TT50, spinal cord and pronephros (lateral). ba, branchial arches; cns, central nervous system; dpn, distal pronephros; e, eye; hb, hindbrain; l, lens; llp, lateral line primordium; mbs, midbrain stripe; nr, neural retina; ov, otic vesicle; pfb, pectoral fin buds; rb5, rhombomere 5; sc, spinal cord, sm, skeletal muscles; y, yolk. Scale bar, 100 µm.

Compatibility of TRAP technology with the Gal4/UAS system:(A): Zebrafish embryo from the control cross Tg[Rx3:Gal4] x Tg[UAS:RFP] showing RFP expression in the developing retina (arrow) at 24 hpf. (B): zebrafish embryo derived from a Tg[Rx3:Gal4] × Tg[UAS:TRAP] cross showing eGFP-rpl10a expression at 24 hpf in the retina (arrow). l, lens; y, yolk. Scale bar = 100 µm.