Extended Data Fig. 4: Generation of trans-Tango transgenic zebrafish.
From: Transsynaptic labeling and transcriptional control of zebrafish neural circuits
Heterozygous fish bearing transgenes for trans-Tango components and Tg(QUAS:mApple-CAAX, he1.1:mCherry) were mated with the TgBAC(pt1fa:Gal4-VP16)jh16 driver line and resultant embryos injected with plasmids for the other trans-Tango reagents and Tol2 RNA. a, RNA in situ hybridization indicates that the expression of UAS:sGCG-ICAM(1235)-Nrxn1b resembles the transcription pattern of the endogenous ptf1a gene99. b,c, Both the Tg(elavl3:hArr-TEV, he1.1:YFP)cd30 (b) and Tg(elavl:hGCGR-TEVcs-QF, he1.1:CFP) (c) stable lines are expressed broadly throughout the CNS. d,d″, Tg(10xUAS-E1B:sGCG-ICAM(1235)nrxn1b, cryaa:mCherry) larvae showed trans-Tango labeling in the hindbrain (n = 257/312) consistent with that observed when the ligand is introduced by plasmid injection. e,e″, Tg(elavl3:hArr-TEV, he1.1:YFP) larvae had similar trans-Tango labeling in the hindbrain (n = 132/164). f,f″, mApple-CAAX labeling was not observed in the hindbrain (n = 0/276) with the receptor line Tg(elavl:hGCGR-TEVcs-QF, he1.1:CFP) or when receptor plasmid was injected at a sub-threshold concentration (10 ng μl−1) along with the ligand and arrestin–TEV plasmids (25 ng µl−1; n = 25; g,g′). h,h′, However, the same concentration of receptor plasmid was effective at producing extensive mApple-CAAX labeling in the hindbrain (n = 21/25) when injected into embryos bearing Tg(elavl:hGCGR-TEVcs-QF, he1.1:CFP) along with the ligand and arrestin–TEV plasmids (25 ng µl−1). Scale bars, 20 µm for a–c and 50 μm for d–h. Images are representative of five independent experiments for each stable line.
