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Fig. 4 CNS4 contains pre-thalamic enhancer activity and relies on a zic binding site.
(A-D) Double in situ hybridization results indicate CNS4 contains pre-thalamic enhancer activity. ZLI (zona limitans intrathalamica) marker shha and thalamic marker dbx1a are stained in dark blue, reporter egfp stained in red. CNS4 driven EGFP expression abuts anteriorly to the ZLI and thalamus in the 24hpf (A-B) and 48hpf (C-D) stage embryos. (E-H) The third fragment of the four subdivisions of the CNS4 sequence contains pre-thalamic enhancer activity. (I) Multiple sequence alignments of the CNS4 region in the five teleost species. The identified zic binding site is highlighted in blue. (J) Mutation of the 8bp zic binding site in the prethalamic enhancer abolished its activity. Lateral view of the forebrain regions of embryos at 24 hpf (A-B, E-H, and J) or 48 hpf (C-D), anterior to the left. Scale bar: 50µm.