Fig. 2

CNE3 balances transcriptional activation and repression to establish the Pax3 expression domain.

(A) TCFsiam transgenic zebrafish assessed at 10 hpf reveal activated Wnt signaling in the posterior neural plate and tailbud (bd). (A′) Transverse sections of the posterior neural plate demonstrate that Wnt pathway activity is not restricted in the medio-lateral axis of the tissue, whereas Pax3/7 is expressed laterally. (B–C) Wnt signaling is maintained within the neural tube up to 18 hpf, following which it rapidly declines and is found only in the dorsal most row of cells within the neural tube at 24 hpf. (D) Matrix indicating the conservation of CNE3-Motif5 across 12 vertebrate genomes. (E, E′) Zebrafish injected with a CNE3 reporter rarely contain labeled cells within the dorsal spinal cord of transient transgenic embryos at 24 hpf (n = 4/33) and the reporter is not active in the chick neural tube at E3 (F) (n = 0/7). (G, G′) CNE3Motif5Del::Citrine transgenics exhibit an increase of enhancer activity across the DV axis of the spinal cord, compared to controls (n = 28/32, p<0.0001). (H) Furthermore, deletion of Motif5 results in the ectopic induction of CNE3 activity in chick embryos at E3 (n = 4/4, p = 0.003). (I) Schematic depicting the organisation of motifs within CNE3 and the mutation induced within the conserved HD binding site in Motif5. (J, J′) Mutation of the HD binding site in Motif5 increases CNE3 activity in progenitors within the zebrafish spinal cord, compared to controls (n = 21/21, p<0.001). (K) Chick embryos electroporated with CNE3M5HDMut DNA exhibit ectopic enhancer activity in the developing neural tube (n = 3/4, p = 0.0242). (L) EMSAs performed using a DNA probe spanning Motifs 3–5 of CNE3 and chick spinal cord nuclear extract. Complexes formed with nuclear extract are indicated (arrows, compare Control to Probe lanes). Addition of an Nkx6.1 antibody creates a slower migrating DNA/protein complex than controls, whereas addition of FoxA2 antibody abrogates complex formation. Both results indicate that these proteins are able to bind CNE3. Addition of Dbx1, Dbx2, Pax6 and Nkx6.2 antibodies have a minor effect on the motility of EMSA complexes. (M, M′) Consistent with binding and mutagenesis studies, the electroporation of Nkx6.1 in the chick neural tube is sufficient to repress endogenous Pax3 protein expression in chick embryos (n = 11).