Non-coding conserved elements drive mosaic gfp expression in the midline of transient transgenic zebrafish embryos. (A) Zebrafish embryo injected with control gfp-reporter construct, containing a minimal - 0.563 kilobase (kb) zebrafish shh promoter without cis-regulatory motif. (B to H) Embryos were injected with the gfp-reporter 0.563shh:GFP fused to the non-coding conserved sequences arx A7 (B), id1 A8 (C), nkx2.2b A21 (D), sox9a E1 (E), ctgf up1 (F), shh ar-C (G) and shh ar-A (H), respectively. arx A7 (B) and nkx2.2b A21 (D) drove expression in the floor plate while id1 A8 (C), sox9a E1 (E), ctgf up1 (F), shh ar-C (G) and shh ar-A (H) mediated gfp expression in the notochord at 24 h post-fertilization (hpf). Embryos are oriented anterior to the left and dorsal up. Scale bar = 250 μm.

Fine-mapping of the notochord regulatory sequences in ctgf up1. The left panels show gfp expression in the midline of 24 hpf zebrafish embryos. Right panels are schematic representations of the ctgf wild-type promoter (A) and mutant constructs (B to H) cloned upstream of the TATA-box of the ctgf gene and gfp-reporter cassette. Positions give base pairs upstream of the ATG. The transversions introduced to mutate the C1 and C2 elements are indicated. Embryos are anterior left and dorsal up. Scale bar = 250 μm.

Linker scanning analysis of the sox9a E1 enhancer identifies a subregion as necessary for notochord expression. (A) Reporter constructs designed to test the activity of the E1 enhancer. A 348 bp fragment containing a 135 base pair (bp) conserved core (E1 core) of the sox9a E1 enhancer was cloned downstream of the 2.4 kb shh promoter and gfp reporter gene (- 2.4shh:GFP). Clusters of 20 bp transition mutations are represented by black boxes. Numbers represent the positions relative to the sox9a ATG. (B-M) Lateral view of 26 h old zebrafish embryos, injected with the constructs RC0 to RC11. Expression in the notochord (n) is abolished in embryos injected with the constructs RC6 to RC9. Remaining anterior floor plate (fp) expression of - 2.4shh:GFP serves as an injection control. Anterior to the left and dorsal up. Scale bar = 250 μm.

Conserved M1 and M2 motifs are both required to drive gfp expression in the notochord. (A) Embryo injected with the gfp-reporter control, containing 80 bp of the ctgf basal promoter (TATA box). This promoter is inactive without addition of an upstream CRM. (B, C, D) gfp-reporter constructs under the control of M1 and M2 motifs from ctgf up1 (B), sox9a E1 (C) or id1 A8 (D) drive gfp expression in notochord cells. (E, F) Three copies of either the ctgf M1 motif (E) or of the ctgf M2 motif (F) are not sufficient for gfp-reporter expression, indicating that each of the elements alone is not sufficient and that the combination of the two elements specifies notochord expression. Embryos are 26 hpf old and are oriented anterior to the left and dorsal up. Numbers in the schematic drawing to the left indicate positions of fragments relative to the ATG of the respective linked gene. Scale bar = 250 μm.

Foxa2 protein binds to the M1 motif of ctgf up1 and sox9a E1 CRMs. (A) Recombinant Foxa2 binds specifically to M1 of the ctgf up1 CRM. Radioactively labeled oligonucleotide containing ctgf up1 M1 (lane 1) was incubated with Foxa2 protein without competitor oligonucleotide (lane 2), with a 50-fold molar excess of cold ctgf up1M1 oligonucleotide (lane 3), or with an oligonucleotide that contained a mutated M1 sequence (lane 4). Cold M1 competitor abolished the shifted band (lane 3), whereas mutated oligonucleotide did not (lane 4). Radioactively labeled mutated ctgf up1 M1 probe alone (lane 5) or in the presence of Foxa2 protein (lane 6) does not show retarded DNA protein complexes. (B) FoxA2 binds specifically to the sox9a E1M1. Radioactively labeled oligonucleotide containing sox9a E1M1 (lane 1) was incubated with FoxA2 protein without competitor oligonucleotide (lane 2), with a 50-fold molar excess of cold sox9a E1 M1 oligonucleotide (lane 3), or with an oligonucleotide that contained a mutated sox9a E1 M1 sequence (lane 4). Cold M1 competitor abolished the shifted band (lane 3), whereas mutated oligonucleotide did not (lane 4). Radioactively labeled mutated sox9a E1 M1 probe alone (lane 5) or in the presence of Foxa2 protein (lane 6) does not show retarded DNA protein complexes.