Duel-florescence transgenic analysis of a SHH enhancer SBE2 where a C>T change in a Six3 binding site has been identified in a patient with holoprosencephaly.

(A) Regulatory landscape of SHH, depicting the location of the SBE2 enhancer. The sequence conservation plot on the right shows the low level of sequence conservation for SBE2 in the zebrafish genome. (B) SHH-SBE2 enhancer-driven F1 reporter transgenics. The wild-type allele Wt(C) drives expression in the rostral and caudal hypothalamus (long green or red bar) independent of the associated fluorophore (eGFP or mCherry). Expression from the mutant allele, Mut(T), is retained in the caudal hypothalamus (short green or red bar) but lost in the rostral hypothalamus (short green or red bar). Additional expression of the SBE2 Wt(C) allele in the rostral forebrain of later stage (96hpf) zebrafish embryos is also lost by the Mut(T) allele. (C) RNA in situ hybridisation analysis of shha and shhb expression at 48hpf and 96hpf of zebrafish embryonic development. The reporter gene expression pattern driven by Wt(C) allele significantly overlaps with the shha expression domain in the hypothalamus. (D) Morpholino knock-down of Six3 in SHH-SBE2 transgenic embryos mimics the effect of the Six3 binding site mutation in SBE2. F2 embryos bearing both SHH-SBE2 Wt(C) and Mut(T) alleles injected with either control morpholino (Control MO) or morpholino against both six3A and six3B (six3 AMO). Upon knockdown of six3, the hypothalamus expression driven by the Wt allele shrinks to overlap completely with the expression driven by the mutant allele (bearing a mutant six3 binding site). Wt: wild-type; Mut: mutant; hpf: hours post fertilization; MO: morpholino, RH: Rostral hypothalamus; CH: Caudal hypothalamus; rFB: Rostral forebrain.

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.

Dual-fluorescence transgenic analysis of IRF6 enhancer MCS-9.7 where a SNP (rs642961, G>A) in a TFAP2A (AP2α) binding site has been associated with cleft lip.

(A) Regulatory landscape of IRF6, depicting the location of MCS-9.7 enhancer. The conservation plot on the right shows the absence of sequence conservation for MCS-9.7 in the zebrafish genome. (B) IRF6-MCS-9.7 enhancer-driven F1 reporter transgenics. The Wt(G) allele drives expression in the first pharyngeal arch (PA1) (arrow) and in the developing ethmoid plate (EP) (curved arrow). Mut(A) has lost EP expression but maintains PA1 expression. (C) RNA in situ hybridisation analysis of zebrafish irf6 at 72hpf showing overlap of the reporter gene expression domain driven by Wt(G) allele with the endogenous irf6 expression pattern in the developing jaw. Wt: wild-type; Mut: mutant; hpf: hours post fertilization.

Characterisation of novel enhancers from the SOX9 genomic region with PRS-associated point mutations: spatial loss of expression.

(A) Regulatory landscape of SOX9, depicting the location and sequence conservation across evolution for the novel SOX9 enhancers. (B) p300-PK19: Lateral and ventral views of F1 dual-fluorescence reporter transgenic embryos at 48 hpf. The Wt(G) allele drives expression in the region around the oral cavity (OC), pharyngeal arches (PA2-5), otic vesicle (OV), ciliary margin zone (CMZ) of the eye, and midbrain (MB). The Mut(A) allele has lost expression in the region around the oral cavity and pharyngeal arches (curved white arrow) and the midbrain (white arrow). (C) hoc-CNE-A: F1 dual-fluorescence reporter transgenic embryos at 72 hpf and 96 hpf. At 72 hpf the Wt(C) allele drives expression in the olfactory placodes (OP), and in the palatoquadrate (PQ), ceratohyal (CH), ceratobranchial (CB) (weak) and hyosymplectic (HS) cartilages. The Mut(T) allele has lost PQ, CH, CB and HS expression; OP expression is retained. At 96 hpf the Wt(C) allele drives expression in PQ, CH, CB. The Mut(T) allele has lost CH and CB expression, but retains reduced PQ expression. (D) RNA in situ hybridisation analysis of zebrafish sox9a at 48–96hpf showing overlap of reporter expression driven by the p300-Pk19 Wt(G) and hoc-CNE-A Wt(C) alleles with the endogenous sox9a expression pattern in the developing jaw. Wt: wild-type; Mut: mutant; hpf: hours post fertilization.

New enhancers from the SOX9 region with PRS-associated single nucleotide variants.

(A) p300-PK17, spatiotemporally altered expression: F1 dual-fluorescence reporter transgenic embryos shown at 48, 72 and 96 hpf. At 48 and 72 hpf the Wt(G) allele drives expression in the region around the oral cavity (OC) (white arrowhead). At 96 hpf Wt(G) drives expression in the palatoquadrate (PQ) (curved white arrow) and Meckel’s cartilage (MC) (open arrow). The Mut(A) allele is able to drive OC expression at 48 hpf, but this is lost by 72 hpf and at 96 hpf no PQ and MC expression is observed. (B) hoc-CNE-D, tissue-specific CRE with unaltered expression: F1 dual-fluorescence transgenic embryos at 72 and 96 hpf. The Wt(T) and the Mut(C) alleles both drive expression in olfactory placode (OP), brain (BR), and ceratobranchials (CB). (C) p300-PK22, craniofacial CRE with unaltered expression: F1 dual-fluorescence transgenic embryos at 72 hpf. The Wt(A) and Mut(C) allele both drive expression in the region around the oral cavity (OC). (D-E) RNA in situ hybridisation analysis of zebrafish sox9a and sox9b at 72hpf and 96hpf, showing overlap of reporter gene expression driven by the p300-Pk17, hoc-CNE-D and p300-Pk22 elements with the endogenous sox9a and sox9b expression pattern in the developing jaw. Wt: wild-type; Mut: mutant; hpf: hours post fertilization.

Wild type and variant CRE driven transgene expression patterns in F1 embryos obtained from multiple independent stable transgenic lines.

Dual reporter fluorescence in transgenic F1 embryos from multiple additional independent lines for the SHH-SBE2 (A), SHH-ZRS (B), PAX6-SIMO (C), p300-Pk19 (D) and p300-Pk17 (E) elements. These lines are independent from the ones shown in the main text. The expression sites that are consistent between the multiple transgenic lines for each of the CREs are marked. RH: rostral hypothalamus; CH: caudal hypothalamus; PF: pectoral fin; L: lens; CMZ: ciliary margin zone; OV: otic vesicle; PA2-5: pharyngeal arch 2–5; OC: oral cavity; PQ: palatoquadrate; MC: Meckel’s cartilage; Wt: wild-type; Mut: mutant; hpf: hours post fertilization.

SHH-ZRS in multiple founders.

SHH-ZRS enhancer-driven reporter expression is shown at 72 hpf (A) The Wt(G) allele is shown driving eGFP expression in a restricted region of the developing pectoral fin (PF) in two independent founders (1 and 2). (B) The Wt(G) allele is shown driving mCherry expression in a restricted region of the developing pectoral fin (PF) in four independent founders (1–4). Consistent reporter gene expression was detected only in the pectoral fin in all independent founders shown.