Fig. 2

Screen for floor plate enhancer activity in the netrin1 locus. (A) Floor plate enhancer activity maps to the 898-bp fragment 6 located in intron 2 of the netrin1 locus. Scheme illustrating the position of fragments tested in the co-injection assay. Fragments were injected together with –1.1netrin1:lacZ, and embryos were scored for floor plate expression by β-galactosidase staining at 24 hpf. Only fragments C, 3, 4, and 6 gave significant numbers of β-galactosidase-positive cells in the floor plate (FP). (B, C) Embryos were analysed for GFP expression between 36 and 48 hpf. (B) Transient expression of the promoter –1.1netrin1:gfp. Embryos were injected with the promoter construct and analysed for GFP expression. (C) The 898-bp floor plate enhancer (FPE, fragment 6) downstream of the –1.1netrin1:gfp cassette (-1.1netrin1:gfp/FPE) directs gfp expression in the floor plate (arrow, f) and the hypochord (arrowhead), h. Expression in cells of the notochord was also noted in these transiently expressing embryos. (D, E) Transient expression of the –37Tk:gfp minimal promoter alone (D) or the 898-bp enhancer (E) inserted downstream of the thymidine kinase promoter and the gfp reporter gene (-37Tk:gfp/FPE) at 24 hpf. Insertion of the enhancer leads to expression in the floor plate (arrow) and the hypochord (arrowhead). Abbreviations: f, floor plate; h, hypochord. The scale bar represents 100 μm (B–E).