Fig. 4

Cre-mediated recombination of Gt(foxd3-citrine)^ct110a results in expression of a mutant FoxD3-mCherry fusion protein. (A) Schematic diagram of Cre-loxP recombination in Gt(foxd3-citrine)^ct110a zebrafish line. The FlipTrap construct contains inverted mCherry (red box) and polyA (pA) sequences, flanked by heterotypic loxP (light gray) and loxPV (dark gray) sites. In embryos injected with cerulean-cre mRNA, recombination of the loxP sites results in deletion of citrine and correct orientation of mcherry-pA. Splicing of foxd3 cryptic splice donor site with mcherry splice acceptor site (SA) produces a mutant FoxD3ΔFkd-mCherry fusion protein, expressed under foxd3 endogenous regulation. (B–K) Expression of mcherry co-localizes with endogenous foxd3 in Gt(foxd3-mcherry)^ct110R embryos, whereas Tg(foxd3:GFP) line recognizes a subpopulation of NCCs. (B–G) Fluorescent whole mount in situ hybridization in Gt(foxd3-mcherry)^ct110aR embryos at 22 hpf. Expression of mcherry (B) is detected in the branchial arches, in the same distribution as endogenous foxd3 (C and D). High-resolution confocal imaging of mcherry (red; E and G) and foxd3 (green; F and G) shows co-expression in the branchial arches, as shown in the inset (E′, dashed line). (H–K) Confocal imaging of double transgenic Gt(foxd3-mcherry)^ct110aR; Tg(foxd3:GFP) embryo at 18-somite stage. Expression of ct110aR (red) allows visualization of early NCCs associated with the neural tube (K) and NC in the branchial arches (H). In contrast to expression of FoxD3-mCherry, which comprehensively labels neural crest cells, expression of GFP (green) driven by a 14 kb promoter is heterogeneously distributed (H and J) and shows restricted expression in the branchial arches at 18ss (J and K). Scale bars: 50 μm.