Fig. 3

Dual-fluorescence transgenic analysis of established CREs with disease-associated point mutations.

(A) SHH-ZRS. (a) The regulatory landscape of SHH, depicting the location of ZRS enhancer. The conservation plot on the left shows the low level of sequence conservation for ZRS in the zebrafish genome. (b) SHH-ZRS enhancer-driven reporter expression is shown at 72 hpf and 96 hpf. The top row shows the Wt(G) allele driving both mCherry and eGFP expression to equal measure in a restricted region of the developing pectoral fin (PF), coinciding with the expression domain of both shha and shhb in the developing pectoral fin (c). The Mut(A) allele drives expression at an additional site on the opposite edge of the fin. The ectopic expression (arrowheads) is increased by 96 hpf. The double-headed arrow indicates the anterior (a)—posterior (p) axial plane of the developing pectoral fin. (B) PAX6-SIMO. (a) The regulatory landscape of PAX6, depicting the location of SIMO enhancer. The conservation plot on the right shows the presence of sequence conservation for SIMO in the zebrafish genome. (b) PAX6-SIMO enhancer-driven reporter expression is shown at 72 hpf. In contrast to the Wt(G) element, and irrespective of the fluorophore used, the Mut(T) allele consistently fails to drive reporter gene expression in the developing lens (L). (c) RNA in situ hybridisation analysis of zebrafish pax6a at 72hpf showing the endogenous pax6a expression pattern in the developing eye with strong retinal expression and weaker lens expression overlapping with the reporter domain driven by the Wt(G). Wt: wild-type; Mut: mutant; hpf: hours post fertilization.