A PAC clone comprising the netrin1 locus drives expression of reporter gene in the ventral neural tube. (A) Map of the netrin1 PAC clone. Blow-up shows the promoter region and the position of the green fluorescent protein (gfp) tag brought under control of the netrin1 regulatory region by insertion into the first coding exon. (B) Embryo (24 hpf) expressing the gfp-tagged netrin1 PAC clone. Mosaic expression of GFP can be detected in cells of the floor plate (arrows) and the hypochord (arrow heads). (C) Embryo (24 hpf) hybridised to the netrin1 antisense probe. (B, C) Lateral views, anterior left and dorsal up. The scale bar represents 100 μm in (B) and (C). Abbreviations: N, NotI; B, BamHI; Bs, BssHII; Kb, kilobase pairs; ATG, transcription start; f, floor plate; h, hypochord.

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).

Stable expression of the –1.1netrin1:gfp/FPE transgene. (A) Expression of the transgene in floor plate (f) and hypochord (h) of a 32-hpf embryo. (B, C) Whole-mount in situ hybridisation of transgenic embryos with gfp antisense RNA at the 32-hpf (B) and one-somite stages (C). (D, E) Comparison of expression of netrin1 (D) and gfp (E) mRNA in the head of 24-hpf transgenic embryos. Note that, unlike netrin1 (D), the transgene (E) is not expressed in the ventral forebrain of embryos. (A, B, D, E) Lateral views, anterior left and dorsal up. (C) Dorsal view, anterior up. Abbreviations: f, floor plate; h, hypochord. Scale bar represents 50 μm (A, B, D, E) and 100 μm (C).

The zebrafish –1.1netrin1:lacZ/FPE transgene is expressed in the floor plate (arrow) of mouse (A) and chicken (B) embryos. Transverse section through the spinal cord (thoracic level) of mouse and chicken embryos at 9.5 dpc and at stage 18, 19 HH, respectively. Scale bar represents 25 μm.

The Shh signalling pathway does not control the activity of the –1.1netrin1:gfp/FPE transgene. (A, B) Expression of endogenous netrin1 mRNA in an uninjected control (A) and a shh mRNA-injected embryo (B). Ectopic expression of shh causes expansion of endogenous netrin1 expression in the neural tube (arrowheads). (C, D) Expression of gfp mRNA in control uninjected (C) and shh-injected transgenic embryo (D). In contrast to the endogenous netrin1 gene, the –1.1netrin1:gfp/FPE transgene does not respond to ectopic shh. (E, F) Transgene expression in a wildtype control embryo (CO, E) and Smu^b641 mutant background (F). –1.1netrin1:gfp/FPE expression is not significantly different in the floor plate of wildtype and smu mutant embryos. Orientation of embryos: anterior left, dorsal up. Abbreviations: f, floor plate; h, hypochord; CO, control. Scale bar represents 100 μm.

The Cyc (Nodal) signal controls the activity of the netrin1 transgene. (A, B) netrin1 (A) and –1.1netrin1:gfp/FPE (B) expression in the floor plate is absent in cyc mutants (blue arrowhead) while the hypochord expression is normal (black arrowhead, h). (C, D) Cyc misexpression induces ectopic expression (white arrowhead, e) of netrin1 (C) and gfp (D). Orientation of embryos: lateral views of the trunk at 24 hpf, anterior left and dorsal up. Abbreviations: e, ectopic floor plate; f, floor plate; h, hypochord. Scale bar represents 25 μm.

foxA2 is required for floor plate formation. Left panels show expression of GFP (A), netrin1 (C, E), shh (G, I), and collagen2a1 (K) in uninjected control embryos. Black arrow, expression in the floor plate (GFP, netrin1, shh, col2a1); black arrowhead, expression in the hypochord (GFP, netrin1, col2a1); and red arrowhead, expression in the notochord (shh, col2a1). Right panels represent expression of the same markers in foxA2 morpholino-injected embryos. Note that expression of GFP (B), netrin1 (D, F), shh (H, J), and collagen2a1 (L) is diminished or totally absent (blue arrowhead). Expression of these markers is normal in the notochord (red arrowhead: shh, col2a1), hypochord (black arrowhead: GFP, netrin1, col2a1), and ventral brain (shh, netrin1). (M) In FoxA2 knockdown embryos, the characteristic columnar shape of floor plate cells seems to be maintained (M, green arrowhead). These cells do not express col2a1 even in strongly overstained embryos (M). Abbreviations: CO, control; MO, foxA2 morpholino-injected embryo. Scale bar in (L) represents 100 μm.

foxA2 mimics gain-of-function phenotypes of Cyc/Nodal signals and rescues floor plate in cyc (Nodal) signalling-deficient embryos. Misexpression of foxA2-ER causes ectopic expression of netrin1 (A), gfp (B), and shh (C) in the neural tube of zebrafish embryos (white arrowheads, e). (D, F) netrin1 and shh expression pattern in oep mutants. Note that the expression in the ventral neural tube is absent (blue arrowhead) while the hypochord (black arrowhead, h) and notochord (red arrowhead, n) express normally netrin1 and shh, respectively. (E, G) Injection of foxA2-ER mRNA into oep embryos rescues netrin1 (E) and shh (G) in the neural tube at 24 hpf. Orientation of embryos: anterior is left and dorsal up. Abbreviations: e, ectopic floor plate (white arrowhead); f, floor plate (black arrow). Scale bar represents 50 μm (A–C) and 25 μm (D–G).